Photoelectrochemical (PEC) sensing platforms demonstrate outstanding performances towards enzyme-free glucose detection, and exploring glucose reactive electrodes with efficient active sites and enhanced carrier transport/separation behavior would be beneficial for PEC glucose detection. Herein, we fabricated two-dimensional Cu3(PO4)2 nanosheets as the photoactive material for glucose detection. The morphology and structural characterizations prove the existence of balanced Cu0–Cu+ active sites which has been regarded as the key factor on improving PEC performance for fast glucose detection. The photocurrent of Cu3(PO4)2 nanosheets is three times that of Cu3(PO4)2 bulk in the absence of glucose, and the response performance is increased by about 9 times compared to Cu3(PO4)2 bulk at 50 µM glucose. Under light conditions, the limit of detection (LOD) as low as 17.68 µM at low concentration of 20–100 µM, and LOD of 131.69 µM at high concentration of 100–1000 µM can be achieved. This work might provide the basic understanding and new opportunities in applying two-dimensional Cu3(PO4)2 nanosheets for glucose detection.