Butyrylcholinesterase (BChE) can modulate the expression level of cholinesterase, which emerges as an important clinical diagnose index. However, the currently reported assays for BChE are suffering from the problem of interferences. A ratiometric fluorescence assay was developed based on the MnO2 nanosheet (NS)-modulated fluorescence of sulfur quantum dots (S-dots) and o-phenylenediamine (OPD). MnO2 NS can not only quench the fluorescence of blue emissive S-dots, but also enhance the yellow emissive OPD by catalyzing its oxidation reactions. Upon introducing BChE and substrate into the system, their hydrolysate can reduce MnO2 into Mn2+, leading to the fluorescence recovery of S-dots and failure of OPD oxidation. BChE activity can be quantitatively detected by recording the change of fluorescence signals in the blue and yellow regions. A linear relationship is observed between the ratio of F-435/F-560 and the concentration of BChE in the range 30 to 500 U/L, and a limit of detection of 17.8 U/L has been calculated. The ratiometric fluorescence assay shows an excellent selectivity to acetylcholinesterase and tolerance to various other species. The method developed provides good detection performances in human serum medium and for screening of inhibitors.