The invention discloses a photo-electrochemical diethylstilbestrol biosensor constructed based on a boron-doped poly-metallic oxide as well as preparation and application thereof. In a preparation method, firstly, a two-dimension nano material g-C3N4 is modified on ITO conductive glass by adopting an electro-deposition method; then, an iron cobalt hydroxide is subjected to in-situ growth by adopting a hydrothermal method, and further, a working electrode modified by a g-C3N4-loaded boron-doped iron cobalt oxide Bi@NixFe1-xO3 / g-C3N4 is obtained; finally, a diethylstilbestrol antibody is loaded by utilizing good biocompatibility and large specific surface area of the material. During detection, as the boron-doped iron cobalt oxide Bi@NixFe1-xO3 / g-C3N4 can catalyze L-ascorbic acid-2-trisodium phosphate AAP, L-ascorbic acid AA is generated in situ, and further, an electron donor is provided for photoelectric detection; by utilizing the effect of specific quantitative binding of the antibody and an antigen on electronic transmission capacity, the photocurrent strength is correspondingly reduced, and the construction of a photoelectric sensor for detecting diethylstilbestrol by adopting an unmarked photo-electrochemical method is finally realized.