A signal off type photoelectrochemical (PEC) aptasensor was constructed for the sensitive detection of chloramphenicol based on CuO nanosheets (CuO NSs) and gold nanoflowers (Au NFs) modified indium tin oxide (ITO) electrode. CuO NSs was p-type photoactive semiconductor materials, which had narrow band gap and high absorption rate of visible light. After combining with Au NFs, the composite CuO NSs@Au NFs produced a strong cathodic photocurrent for the excellent electron conductivity, large specific surface area and Surface Plasmon Resonance effect (SPR) of Au NFs. At the same time, Au NFs provided a binding site for aptamer, which greatly improved the sensitivity of the constructed sensing platform. After the target chloramphenicol (CAP) was specifically captured by the aptamers on the electrode, the formed bioconjugates seriously hindered the transfer of electrons and the absorption of light and caused a reduced photocurrent. For the detection of CAP, this signal off type PEC aptasensor had a linear range of 5 pM-300 nM and a limit of detection of 49.9 fM (S/N = 3) with good stability, selectivity and reproducibility. Meanwhile, the method had satisfactory recovery results in the detection of CAP in real-life samples such as milk, tap water, honey and chloramphenicol eye drops.