Despite its excellent efficiency in natural photosynthesis, the utilization of photosystem II (PSII)-based artificial photoelectrochemical (PEC) systems for analytical purposes is hindered due to the low enzyme loading density and ineffective electron transfer (ET) processes. Here, we present a straightforward and effective approach to prepare a PSII-based biohybrid photoanode with remarkable photoresponse, enabled by the use of a hierarchically structured inverse-opal tin oxide (IO-SnO 2 ) electrode combined with gold nanoparticles (Au NPs). The porous, carbon-containing IO-SnO 2 structure allows for a high density and photoactivity loading of PSII complexes, while also providing strong electrical coupling between the protein film and the electrode. A new electron transfer pathway mediated by Au NPs was identified at the protein-electrode interface, which efficiently shuttles the photogenerated electrons from the enzyme to the IO-SnO 2 electrode. Furthermore, the PEC response of the electrode was significantly enhanced by the surface plasmon resonance (SPR) effect of Au NPs. Upon light irradiation, this PSII-based photoanode exhibited an impressively high and stable photocurrent output, which was utilized to fabricate an aptasensor for 17β-Estradiol (E2) detection. Under optimal conditions, a detection limit of 0.33 pM was obtained, along with a broad detection range from 15 pM to 30 nM. The applicability of the aptasensor was assessed by measuring E2 in water and urine samples, demonstrating its feasibility in environmental monitoring and clinical tests.