In this study, we suggested first analytical sensor and especially electrochemical sensor for simultaneous determination of Cholesterol, ascorbic acid and uric acid. Display omitted In this article, we report the simultaneous determination cholesterol (CL), ascorbic (AA) acid and uric acid (UA) at a carbon paste electrode (CPE) modified with copper oxide decorated reduced graphene (CuO-rGR), with 1-methyl-3-octylimidazolium tetrafluoroborate (1M3OIDTFB) as a binder. The electrode, CuO-rGR/1M3OIDTFB/CPE, showed remarkable sensitivities towards the determination of the analytes, and well defined and clearly separated oxidation peaks were obtained during their simultaneous analysis in a buffer solution at pH 7.4. The differences observed between their peaks potentials are as follows: 430 mV (between CL and AA), 270 mV (between AA and UA) and 700 mV (between CL and UA). The morphologies and structure properties of the CuO-rGR were investigated by FESEMD and EDAX methods. The CuO-rGR/1M3OIDTFB/CPE displayed linear response in the concentration ranges 0.04–300.0 μM, 0.04–240.0 μM and 0.4–400.0 μM for CL, AA and UA with the detection limits 9.0 nM, 9.0 nM and 0.08 μM, respectively. The CuO-rGR/1M3OIDTFB/CPE displayed high performance for the determination of CL, AA and UA in real samples.