The sensitive, selective, and accurate analysis of kanamycin (KAN), a common aminoglycoside antibiotic, is vital to ensure human health and food safety. Here, a well-designed photoelectrochemical (PEC) KAN aptasensor was proposed based on the synergistic strategy by Schottky junction and sensitization. A Schottky junction of CuO/Pd nanocomposite was used as a PEC substrate to immobilize an aptamer. The Schottky barrier not only provided an electron-transfer irreversible passage from CuO to Pd shell but also generated a local surface plasmon resonance between CuO and Pd shell, thus improving the efficient charge separation and light absorption. Complementary-DNA-functionalized CdS quantum dots (CdS QDs) were introduced onto the surface of CuO/Pd via hybridization of the target aptamer to improve the response of the PEC aptasensor further. When CdS was close to Pd shell, the Fermi energy level of CdS was readjusted under the action of the sensitization. The photogenerated hot electrons on the conduction band of CdS were transferred into Pd by a powerful interfacial electric field. Upon blue-light irradiation, the change of photocurrent depended upon the amount of stored negative charge on the surface of the Pd shell and stored positive charge on the valence band of CdS before and after incubation with KAN. Using the photocurrent change as a response signal for the quantitative detection of KAN, the PEC aptasensor exhibited a low detection limit of 20 pM with a wide linear range of 0.1–500 nM. Thus, the developed PEC aptasensor has great potential for applications in various fields.