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Aflatoxin B1 (AFB1) is the most toxic mycotoxin, is widely found in foods and animal feeds, and can pose a serious threat to our lives. A label-free photoelectrochemical (PEC) immunosensor was fabricated for the sensitive detection of AFB1. A Z-scheme heterojunction of gold nanoparticles (Au NPs) loaded on graphitic carbon nitride sheet and tungsten trioxide sphere composite (g-C 3 N 4 /Au/WO 3) acted as the highly sensitive platform. The g-C 3 N 4 /Au/WO 3 is capable, not only of immobilizing antibodies via Au NPs, but also enhancing the separation of electron-hole pairs due to its good energy band matching efficiency. The mechanism of photo-generated electron/hole transfer on g-C 3 N 4 /Au/WO 3 was explored using scavengers to eliminate active components. On this basis, an electron transfer pathway for the immunosensor was deduced. The PEC immunosensor displayed a linear concentration range from 1.0 pg mL−1 to 100 ng mL−1 and a low detection limit of 0.33 pg mL−1 (S/N = 3) for AFB1. Good reproducibility, stability, and specificity provide a solid foundation for the practical application of this immunosensor. • A PEC immunosensor was developed based on g-C 3 N 4 /Au/WO 3 for detecting AFB1. • g-C 3 N 4 /Au/WO 3 as Z-scheme heterojunction exhibited excellent photocurrent. • The possible transfer routes of charge carriers were studied by scavengers. • The immunosenor possessed a low detection limit of 0.33 pg mL−1. [ABSTRACT FROM AUTHOR]