The first study reports the development of a reusable, single-step system for the detection of specific substrates using oxidase-functionalized Fe3O4 nanoparticles (NPs) as a bienzyme system and using amplex ultrared (AU) as a fluorogenic substrate. In the presence of H2O2, the reaction pH between Fe3O4 NPs and AU was similar to the reaction of oxidase and the substrate. The catalytic activity of Fe3O4 NPs with AU was nearly unchanged following modification with poly(diallyldimethylammonium chloride) (PDDA). Based on these features, we prepared a composite of PDDA-modified Fe3O4 NPs and oxidase for the quantification of specific substrates through the H2O2-mediated oxidation of AU. By monitoring fluorescence intensity at 587 nm of oxidized AU, the minimum detectable concentrations of glucose, galactose, and choline were found to be 3, 2, and 20 £gM using glucose oxidase-Fe3O4, galactose oxidase-Fe3O4, and choline oxidase-Fe3O4 composites, respectively. The identification of glucose in blood was selected as the model to validate the applicability of this proposed method. The second study follows the first one. Using the catalytic activity of Fe3O4 NPs with AU to detect four kinds of neurotransmitter, such as dopamine, L-DOPA, adrenaline (epinephrine) and noradrenaline (norepinephrine). Because of there is specific interaction between Fe3O4 NPs and catecholamines (CAs), the Fe3O4 NPs will form CAs-Fe3O4 NPs composites in presence of CAs. The CAs on the Fe3O4 NPs surface must shelter the reaction between AU and H2O2, cause the fluorescence to be turned-off. The CAs just like a inhibitor, to inhibit the catalytic activity of Fe3O4 NPs. Therefore, we could use this inhibited system to detect the CAs compound concentration in the real sample.