A novel and simple photoelectrochemical (PEC) bioassay protocol for estradiol was proposed based on in situ generated CdS-enhanced TiO2 film via competitive strategy. The CdS was generated in situ by immediately dropping S(2-) onto the Cd(2+)-functionalized titanium phosphate nanoparticles (TiP@Cd(2+)). The TiO2 photoactive sensing film with countless active sites was obtained by calcination and further explored for estradiol (E2) capture. The TiP@Cd(2+) was used as labels and immobilized through affinity-specific binding with E2 on the surface of the electrode. Greatly enhanced sensitivity was achieved by using porous TiP nanoparticles as carriers to load a large amount of Cd(2+) and further for more CdS production through the S(2-) deposition. What's more, the photocurrent of CdS generated on the electrode surface could be significantly amplified by the coupling of CdS and TiO2, which could enhance the excitation and photo-to-electric conversion efficiency. Through the application of a competitive binding assay, the proposed biosensor showed high sensitivity with a detection limit down to 2pg/mL. This simple and fast PEC E2-sensing approach offers great promise to extend its application for the assay of small molecules of biomedical, food and environmental interest. Additionally, the strategy of employing in situ generated narrow-band gap semiconductors paves a new way for PEC sensing.