DNA walkers have shown superior performance in biosensing due to their programmability to design molecular walking behaviors with specific responses to different biological targets. However, it is still challenging to make DNA walkers capable of distinguishing DNA targets with single-base differences, so that DNA walkers that can be used for circulating tumor DNA sensing are rarely reported. Herein, a flap endonuclease 1 (FEN 1)-assisted DNA walker has been proposed to achieve mutant biosensing. The target DNA is captured on a gold nanoparticle (AuNP) as a walking strand to walk by hybridizing to the track strands on the surface of the AuNP. FEN 1 is employed to report the walking events by cleaving the track strands that must form a three-base overlapping structure recognized by FEN 1 after hybridizing with the captured target DNA. Owing to the high specificity of FEN 1 for structure recognition, the one-base mutant DNA target can be discriminated from wild-type DNA. By constructing a sensitivity-enhanced DNA walker system, as low as 1 fM DNA targets and 0.1% mutation abundance can be sensed, and the theoretical detection limits for detecting the DNA target and mutation abundance achieve 0.22 fM and 0.01%, respectively. The results of epidermal growth factor receptor (EGFR) L858R mutation detection on cell-free DNA samples from 15 patients with nonsmall cell lung cancer were completely consistent with that of next-generation sequencing, indicating that our DNA walker has potential for liquid biopsy.