Yellow fever virus (YFV) virus is an arthropod-borne virus that causes viral hemorrhagic fever in humans in the tropical parts of both Africa and South America. The virus belongs to the family Flaviviridae, of the genus Flavivirus comprising of approximately 70 viruses. It is transmitted to vertebrates by the bite of an infected female mosquito, primarily the Aedes species. It is a re-emerging pathogen with case-fatality rates that can exceed 50% in humans. YFV can cause an acute febrile illness in humans which can progress to severe disease with hepatic and renal failure. The diagnosis of infection and testing of the immune status of vaccinees require reagents that are prepared in biosafety level (BSL) three and four facilities. Therefore the development of a recombinant antigen that does not require BSL three facilities for preparation and is safe to use, would have an important role in a diagnostic laboratory for detecting antibodies in infected individuals and vaccinees. Despite the availability of a live-attenuated efficacious vaccine, it is not recommended for immunocompromised individuals, thus development of new generation vaccines would have important public health implications. Identification and mapping of antigenic regions and viral epitopes is important for development of subunit vaccines and improved diagnostics. Subunit vaccines focusing on antigens that induce a protective immune response provide a safe approach to the development of vaccines against diseases causing severe and frequently fatal haemorrhagic fevers. The aim of this study was to identify immunodominant viral proteins that induce detectable antibody responses that could be used for developing diagnostic assays and to identify linear B cell epitopes on selected viral proteins. The complete open reading frame of the genes encoding the domain III (EDIII) region of the envelope protein, capsid (C) and NS4a proteins of YFV were amplified, from the 17D strain of YFV, by RT-PCR using primers specifically designed from sequence data retrieved from GenBank. Oligonucleotide primers were modified with BamHI and HindIII restriction enzyme sites that facilitated downstream cloning. Each amplicon was cloned into the pGEM®-T Easy cloning vector using T/A cloning. Each gene was rescued from the recombinant plasmid using BamHI and HindIII restriction enzyme sites and ligated into bacterial expression system, pQE-80L vector. In a previous study, the YFV EDIII gene was cloned into pQE-80L and expressed in JM109 Escherichia coli cells however extremely low yields were obtained. In this study the expression levels were improved using different cell lines and optimizing incubation conditions. An insoluble 13 kDa protein was expressed from the construct and confirmed by Western blot analysis. The protein was expressed with a 6 x Histidine tag that was used to facilitate purification using a Ni2+ column under denaturing conditions. Attempts to express the YFV C and NS4a proteins were not successful and expression was abandoned. In an attempt to improve solubility the YFV EDIII gene was excised from the pGEM®-T Easy vector and subsequently cloned into pCold TF bacterial expression vector. A ~65 kDa soluble protein was expressed from the construct and purified under native conditions. The functional activity of the recombinant antigens in ELISA was compared with whole cell lysate antigen prepared from cell cultures infected with YFV. The biological activity of the recombinant YFV pQE-80L-EDIII antigen was confirmed in immunoassays using serum samples from humans vaccinated with YFV vaccine. Positive sera failed to react in ELISA using pCold TF expressed antigen and this antigen was excluded from further assays. A total of 20/24 serum samples from human vaccinees collected at varying stages after vaccination reacted in an ELISA with the recombinant YFV pQE-80L-EDIII protein and 24/24 reacted in ELISA with whole cell lysate antigen. The EDIII region of the envelope protein was shown to be able to differentiate between West Nile Virus infection and YFV infection in a limited number of convalescent horse sera. The recombinant EDIII protein was used to immunize mice. Serum samples collected from the mice reacted against whole cell lysate antigen in ELISA and was shown to have neutralising antibodies using an in vitro neutralisation assay. Hence the EDIII region of the envelope protein likely induces an important protective immune response. Finally, bioinformatics was used to predict possible epitope regions and using peptide libraries spanning predicted sites, one potential epitopic region was identified in the EDIII protein. Putative epitopic and antigenic regions along the length of the C, NS4a and EDIII proteins of each strain were predicted using the BCPREDS and ABCpred software. In conclusion, the EDIII protein, an immunodominant antigen of YFV, prepared in this study has some potential for differentiation of flavivirus antibodies although it lacks sensitivity for routine diagnosis. A potential epitope, TGHGTVVMQ, from amino acid 21 to 29 on the EDIII protein was identified using bioinformatics and was shown to have reactivity against immune sera. The significance of this epitope needs further investigation. Finally the EDIII region of the YFV protein shows potential as a target region for vaccine development as shown for other flaviviruses but which has not previously been published for YFV.