Uracil-DNA glycosylase (UDG) is a crucial repair enzyme, which is considered a reliable biomarker due to its abnormal expression associated with serious diseases. Herein, DNAzyme-powered cascade walkers were proposed for sensitive detection of UDG. The cascade walkers consisted of a fixed walker and a subsequently activated free walker. The fixed walker was constructed on 13 nm AuNPs by loading a fixed walking strand (WS1) and a track strand 1 (TS1). The WS1 contained a DNAzyme sequence, which was pre-locked by a locking strand (LS) with an uracil base. The TS1 inserted an RNA cleavage site and sealed the same DNAzyme. The free walker tracks were conjugated on 25 nm AuNPs by modifying a FAM-labeled track strand 2 (TS2) with an RNA cleavage site. When UDG specifically recognized the LS, the WS1 was released with the assistance of Endo·IV. Then the WS1 continuously cleaved TS1s to drive the fixed walker, thus releasing many sequences containing DNAzyme. The released sequences acted as free walking strands (WS2s) to repeatedly cleave TS2s to power the free walker, which led to fluorescence accumulation. The cascade walkers successfully detected UDG with a limit of 8.5 × 10 -5 U mL -1 . The cascade walkers offer a new method for sensitively analyzing glycosylase.