Sphingomyelinase (SMase) is closely related to diseases like Niemann-Pick disease and atherosclerosis, and the development of a simple method for the assay of SMase activity is very useful to screen new potential inhibitors or stimulators of SMase or biomarkers of disease. Fluorophore-encapsulated nanoliposomes (FENs) are emerging as a new fluorescent probe for sensing the enzymatic activity. In this work, two fluorochromes (cy7 and IR780) were encapsulated into the liposome of sphingomyelin, and therefore, a sphingomyelin-based ratiometric FEN probe for the SMase activity assay was constructed. The probe shows high selectivity and sensitivity to acid SMase with a detection limit of 4.8 × 10-4 U/mL. Sphingomyelin is the natural substrate of SMase; therefore, the probe has native ability for all kinds of SMase activity assays. Moreover, the probe has been successfully applied to the analysis of acid SMase activity in cells and urine samples. As far as we know, this is the first example of a nanoliposome fluorescence method for assaying acid SMase, and the method is biocompatible and much simpler than the existing ones, which might provide a new strategy for developing new methods for other important esterases.