•A novel photoelectrochemical immunosensor for the detection of VP was assembled.•Ho3+ and Yb3+ doping TiO2 improved the photocurrent responsiveness of PEC sensors.•The working electrode based on CdSe QDs sensitized Ho3+/Yb3+-TiO2 was constructed.•PEC sensors had a detection limit of 25 CFU/mL, a detection range of 102–108 CFU/mL.•This study provides a promising rapid and ultrasensitive detection method for VP. A novel photoelectrochemical (PEC) immunosensor based on CdSe quantum dots (QDs) sensitized Ho3+/Yb3+-TiO2 for the detection of Vibrio parahaemolyticus (VP) was assembled. The working electrode was constructed via the layer-by-layer (LBL) method with the Ho3+/Yb3+-TiO2, CdSe QDs, NHS/EDC, antibody of VP (anti-VP), bovine serum albumin (BSA) modified on the surface of the FTO in sequence. Ascorbic acid (AA) acts as an electron donor to combine photogenerated holes in order to provide a stable current system. Ho3+ and Yb3+ co-doping TiO2 broadened the spectral response range of TiO2 to the infrared region and improved the photocurrent responsiveness of TiO2. The PEC immunosensor, with Ho3+/Yb3+ ratio of 1:5, Ho3+/Yb3+-TiO2 of 2 mg/mL and PBS solution of pH 7.4, had an optimal photocurrent responsiveness. Immobilization of anti-VP was by classical NHS/EDC coupling reactions between COOH groups of CdSe QDs and NH2 groups of the anti-VP. The results indicated that PEC immunosensors had a low detection limit of 25 CFU/mL, a wide detection range of 102–108 CFU/mL, high stability, low price, and short detection time. This method could be promising for the rapid and ultrasensitive detection of pathogenic microorganisms in the food.