Photocurrent polarity switchable photoelectrochemical (PEC) sensing has superior accuracy and anti-interference ability to conventional PEC sensing. The development of a novel strategy for photocurrent polarity switchable sensing is of great interest. Herein, a novel strategy for photocurrent polarity switchable sensing is reported by regulating electrostatic interactions between two semiconductor photoactive materials. Hyaluronic acid (HA)-modified CuO nanosheets show a negatively charged surface, which prevents the attachment of CuO nanosheets to negatively charged CdS nanodendrite-modified photoelectrodes because of the strong electrostatic repulsion. In the presence of hyaluronidase (HAase), the specific hydrolysis of HA on the surface of CuO by HAase can yield a positively charged surface, so CuO can be attached to a CdS-modified photoelectrode via electrostatic attraction, leading to photocurrent polarity switching. The photocurrent polarity switchable detection of HAase activity is achieved with an ultralow detection limit of 2 × 10-3 U mL-1 and a wide linear detection range between 0.01 and 100 U mL-1. This work provides a new and effective photocurrent polarity switching strategy for PEC sensing and a simple and efficient method for detecting HAase activity.