In contrast to the unipedal DNA walker, a bipedal DNA walker features a broader walking area and exhibits faster walking kinetics, leading to enhanced amplification efficiency. In this study, we designed a stochastic three-dimensional (3D) bipedal DNA walker, capable of navigating AuNP-based 3D tracks, driven by exonuclease III (Exo III). This detection system enables the linear detection of the non-invasive biomarker apurinic/apyrimidinic endonuclease 1 (APE1) activity across a range of 0 to 120 U per mL, with a detection limit of 0.03 U per mL. The platform not only offers a novel DNA walker for sensitive APE1 detection in cell lysate but also facilitates the precise assessment of NCA's inhibitory effect on APE1. This research holds promise for future screening of other potential APE1 inhibitors.