We report a one-pot synthesis of poly(diallyldimethylammonium chloride)-coated Fe3O4 (PDDA-Fe3O4) nanoparticles (NPs) by the coprecipitation method. The formed PDDA-Fe3O4 NPs were characterized by powder X-ray diffraction and superconducting quantum interference device. The crystalline structure and superparamagnetic property of PDDA-Fe3O4 NPs were found to be similar to those of Fe3O4 NPs, indicating that the presence of PDDA does not affect the formation of Fe3O4 NPs. Moreover, glucose oxidase (GOx) and citrate-capped gold (Au) NPs, having negative charges, can be easily self-assembled at the surface of cationic PDDA-Fe3O4 NPs. Without the requirement of a peroxidise enzyme, the composites of PDDA-Fe3O4 NPs and GOx effectively catalyzes the H2O2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate acid) (ABTS) in the presence of glucose. This colorimetric method enables the analysis of glucose with a minimum detectable concentration that corresponds to 30 μM. We validate the applicability of this method by determining the concentration of glucose in serum samples. In another application, the composites of PDDA-Fe3O4 and Au NPs were shown to be capable of selectively enriching cysteine-containing peptides through the formation of Au-S bonds. The extracted peptides can be easily detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometer. Both applications open up a new way for the synthesis of bifunctional NPs.