Uracil-DNA glycosylase (UDG) is a typical initiator for base excision repair (BER) process. Since aberrant expression of UDG is relevant to a variety of cancers, analysis of UDG activity with high sensitivity and accuracy is of great importance. We reported herein a sensitive and reliable dual-signal bioassay for UDG activity by coupling photoelectrochemical (PEC) and electrochemical (EC) strategies in one probe electrode. The Au/TiO hybrid was used as a matrix to immobilize substrate DNA (sDNA), which modified with AgInS quantum dot (AIS QD) on the terminal. When UDG exist, the base of uracil was eliminated from the sDNA, and the produced apyrimidinic (AP) site could be cleaved by endonuclease IV (Endo. IV) immediately. Under this situation, the PEC labels of AIS QDs were detached from the electrode, resulting in a "signal-off" trend for PEC signal. After assistant DNA (aDNA) was then assembled, the hybridization chain reaction (HCR) was triggered, and EC labels of ferrocene molecules were introduced, producing a "signal-on" trend for EC signal. Besides, as the produced long double-stranded DNA by the HCR had evident steric hindrance, the PEC signal further decreased. Based on this meticulous design, the dual-signal bioassay for UDG activity showed low detection limits of 4.3 × 10 and 1.9 × 10 U/mL with PEC and EC detection, and accurate analysis of UDG activity in living cells was realized. By just changing the recognition site, this sensitive and reliable dual-signal strategy can be extended to diagnose other DNA repair-related enzymes in the real samples.