The ultrafine monodispersed cuprous oxide (Ufm-Cu2O) nanoparticles have been successfully synthesized by a facile wet chemical method using poly-N-vinylpyrrolidone (PVP) as a capping agent. This colloidal solution of Ufm-Cu2O and chitosan (CS) is electrophoretically deposited (EPD) onto the indium tin-oxide (ITO) glass substrate. Thus synthesized nanocomposite has been characterized by X-ray powder diffraction (XRD, similar to 6 nm), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopic techniques. This novel biomedical nanocomposite platform has been explored to fabricate a cholesterol biosensor by immobilizing cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto Ufm-Cu2O-CS/ITO electrode surface. The seed germination tests of these biomaterials (Ufm-Cu2O-CS nanocomposite and ChOx-ChEtUfm-CuO2-CS nanobiocomposite), conducted using the disc diffusion method, reveal strong activity against the common pathogens and crops, indicating biocompatibility of the nanocomposite. Under optimized conditions, the linearity between the current response and the cholesterol concentration has been obtained in the range of 10-450 mg/dL, with detection limit of 15.9 mg/dLcm(-2) and a high sensitivity of 0.895 mu A/(mg/dLcm(-2)). The proposed biocompatible ChEt-ChOx/Ufm-Cu2O-CS/ITO bioelectrode shows fast response time (<5 s), good reproducibility, and long-term stability. This biocompatible biosensor has been used to determine the total cholesterol levels in human serum samples. Investigated antimicrobial activities of bienzyme-functionalized Ufm-Cu2O-CS nanocomposite are the potential platform for biomedical applications.