Acetylcholinesterase (AChE) is an essential serine hydrolase associated with neurodegenerative diseases and serves as a biomarker. In this work, a ratiometric fluorescence strategy for the sensitive assay of AChE activity is developed based on the decomposition of MnO2 by the enzymatic hydrolysis product and DNAzyme-mediated strand cleavage. The sensing system is termed as MnO2/DNAzyme. DNAzyme locked by H2 strands is inactive in the absence of AChE. The fluorescence emission of fluorescein amidite (FAM)-labeled DNA (DNA-F) at 518 nm is quenched by neutral red (NR) and the fluorescence of NR at 632 nm is simultaneously enhanced due to fluorescence resonance energy transfer (FRET). The presence of AChE triggers the hydrolysis of the substrate acetylcholine (ATCh) to enzymatic thiocholine (TCh), which reduces MnO2 nanowires to Mn2+ and releases the attached H1 strands into the solution. The H1 strands hybridize with H2 strands through the strand displacement reaction. Meanwhile, the activated DNAzyme cleaves the RNA nucleotide of the DNA-F signal probe to release FAM. The fluorescence of FAM at 518 nm is thus recovered, corresponding to a decrement of NR emission at 632 nm owing to the blocking of FRET. The fluorescence ratio of F518/F632 serves as a signal readout for the AChE assay within 5 × 10-4-10 U mL-1, with a limit of detection (LOD) of 2.7 × 10-4 U mL-1. The feasibility of this method was demonstrated by the measurement of AChE activity in human blood, which reveals its promising potential in clinical assays.