Abstract In this article, we used the TiO2–graphene–Pt–Pd hybrid nanocomposites (TGPHs) as an enhanced element of the integrated sensing platform for increasing the surface area as well as improving the electronic transmission rate. Subsequently, Au nanoparticles (AuNPS) and cholesterol oxidase (ChOx) were successively self-assembled to TGPHs with high load amount and superior biological activity. The morphology of TGPHs and stepwise fabrication processes were characterized with cyclic voltammetry (CV), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Based on the efficiently catalytic ability of TGPHs and AuNPS, the fabricated biosensor exhibited wide linear ranges of responses to cholesterol in the concentration ranges of 5.0×10−8−5.9×10−4 M, the limit of detection was 0.017 μM (S/N=3). The response time was less than 7 s and the Michaelis–Menten constant ( K m a p p ) )was found as 0.21 mM. The fabricated biosensor was further tested using real food samples egg, meat, margarine and fish oil, showing that the biosensor has the potential to be used as a facile cholesterol detection tool in food and supplement quality control.