Despite the beneficial effects of antibiotics such as chloramphenicol (CAP), they exert some destructive impacts on human health. We designed an electrochemical sensor based on reduced graphene oxide (rGO)/Au/Co2CuS4 nanohybrid for determination of CAP in food and biological samples. The Co2CuS4 was synthesized from binuclear metal-organic framework (CoCu-BDC) through a two-step process. Nanohybrid was characterized by X-ray photoelectron spectroscopy and transmission electron microscopy. The rGO/Au/Co2CuS4 provides more active sites and good electrical conductivity to reduce charge transfer resistance and improve the electrocatalytic activity for determination of CAP. The prepared sensor has a wide linear range from 7 to 141 nM with a limit of detection of 2.5 nM and a limit of quantification of 21.92 nM. It also provided high selectivity and repeatability with a relative standard deviation of 2.6%. Stability studies showed that the electrode has acceptable performance with efficiency of 95% after 33 days.