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This study focuses on preparing surface enhanced fluorescence (SEF) substrates for use in the enhancement of the emission signal of rhodamine B and fluorescein dyes. Fluorescence spectroscopy has been widely utilized owing to its high sensitivity. SEF is a process where the interactions of fluorophores with the localized surface plasmons of metal nanoparticles results in fluorescence enhancement, increased photostability and rates of system radiative decay which leads to a decreased lifetime. One of the most important factors of SEF studies is to provide a uniform distance between fluorophore and metal nanoparticle in a controlled manner / otherwise, F&ouml / rster resonance energy transfer takes place from fluorophore to metal nanoparticle and emission intensity of fluorophore is quenched. The spherical gold and silver nanoparticles were prepared using the well known and straightforward chemical reduction method, in which sodium citrate acted both as a reducing agent and a stabilizer around the formed nanoparticles. Silver and gold were chosen because of their high plasmon field enhancement. Since plasmon field strongly depends on the shape and size of the nanoparticles, the prepared nanoparticles were characterized using absorption spectroscopy and field emission scanning electron microscopy (FE-SEM). Prior to deposition of silver or gold nanoparticles on glass slides, the slides were derivatized by immersing them into an aqueous solution of 3-Aminopropylethoxysilane (APTES). Following derivatization, silver or gold nanoparticles were deposited by immersing the slides into the colloid mixture. Metal nanoparticle coated slides were characterized using absorption spectroscopy and field emission scanning electron microscopy (FE-SEM). Surface enhanced Raman scattering (SERS) measurements were carried out to observe the plasmon efficiency of the deposited nanoparticles. The SERS measurements were repeated for the duration of two weeks in order to check the stability of the plasmon efficiency. In this study, different types of materials (silica, zinc oxide, gold, stearic acid.) were employed as spacers to observe their effects on fluorescence enhancement. Physical vapor deposition (PVD) and Langmuir-Blodgett (LB) film deposition techniques were used for the formation of the spacer within the substrate. Fluorescence enhancement of rhodamine B and fluorescein was observed on the prepared SEF substrates. Obtained enhancement factors indicate that SEF substrates have the potential for sensitivity improvements of fluorescence sensing in many fields.