A novel amperometric non-enzymatic biosensor was developed for cholesterol (CHO) detection based on electropolymerized para-phenylenediamine (p-PD) monomer as a molecularly imprinted polymer (MIP) on the copper foam (CF) modified with platinum (Pt) and copper oxide (CuO) dual-core nanohybrid (CuO/Pt) which was attached on the GCE using carbon glue. UV-Vis spectrophotometry, X-ray diffraction (XRD), grazing XRD (GXRD), energy-dispersive X-ray spectroscopy (EDX), field-emission scanning electron microscopy (FESEM), and Fourier-transform infrared (FT-IR) spectroscopy were utilized to characterize the prepared nanocomposites. The non-enzymatic CHO biosensor showed a detection limit of 0.035 µM, a sensitivity of 157.85 µAµM− 1cm− 2, and a linear range of 0.4-6 µM. Density functional theory (DFT) was used to select the most suitable functional monomer for synthesizing MIP. For the first time, the effect of sensor loading time in cholesterol solution was investigated on the rise of direct oxidation current. Herein, CF/CuO/Pt/CHO-MIP nanocomposite was employed as a selective and suitable catalyst in CHO oxidation due to their high conductivity, large specific surface area, and good electrocatalytic performance. The superior catalytic activity and selectivity introduce the CuO/Pt nanohybrids on the CF surface as a promising nanomaterial for applications in the direct detection of CHO.