A dual-emission fluorescent nanoprobe was successfully constructed through self-assembling CsPbBr3 perovskite nanocrystals (CsPbBr3 PNCs) and tetraphenylporphyrin tetrasulfonic acid (TPPS). Acetylcholinesterase (AChE) is observed to directly quench the green fluorescence of CsPbBr3 PNCs at 520 nm in the absence of an enzyme substrate, but has no significant influence on the red emission of TPPS at 650 nm. The decreased value of the fluorescence intensities ratio at 520 to 650 nm (ΔF520/F650) is proportional to the logarithmic value of AChE activity ranging from 0.05 to 1.0 U/L. The limit of detection is as low as 0.0042 U/L. The relative standard deviation is 3.6% in eleven consecutive measurements of 0.2 U/L AChE. The method exhibits a good anti-interference capacity since it does not respond to most concomitant species. Satisfactory results are acquired for the determination of AChE activity in human serum samples.