As a broad-spectrum tetracycline antibiotic, the overuse of oxytetracycline (OTC) causes antibiotics residues in the environment and seriously threats to human health owing to effective antibacterial properties. Thus, it is particularly important to design a photoelectrochemical (PEC) aptasensor to detect OTC with excellent performance. Herein, we developed a selective and stable PEC aptasensor of OTC on the basis of Co3O4 nanoparticles (Co3O4 NPs)/graphitic carbon nitride (g-CN) heterojunction, used as PEC active materials. The Co3O4 NPs were successfully grown on the g-CN via grinding and calcining mixture of Co3O4 precursors and bulk g-CN. The Co3O4/g-CN heterojunction with improved light utilization and promoted electrons/holes separation capability can exhibit higher PEC signal than that of g-CN. In order to implement the purpose of specific recognition, OTC-aptamer was introduced into modified electrode to construct highly selective PEC aptasensor for OTC determination, which can possess wide linear range (0.01–500 nM) with low detection limit (3.5 pM, S/N = 3). This PEC aptasensor platform with excellent selectivity and high stability can provide a practical application in the field of water monitoring. Display omitted •Co3O4-based photoactive materials was reported for detecting OTC.•The PEC aptasensor for OTC detection possessed low detection limit of 3.5 pM and excellent selectivity.•The PEC aptasensor has been applied in real water samples for OTC detection.