Abstract Carbon dots (CDs) were synthesized from p-aminophenol and ethylenediamine via one-step under mild condition and used as a sensitive fluorescent nanoprobe for the activity determination of N-acetyl-β- d -glucosaminidase (NAG). In this assay, p-nitrophenol was in situ produced from p-nitrophenyl-β-D-N-acetyl-glucosaminide, which was exclusively hydrolyzed by NAG. The UV absorption peak of p-nitrophenol (maximum at 400 nm) overlapped the excitation peak of CDs with maximum wavelength at 415 nm, which caused the fluorescence decline of CDs based on inner filter effect. The activity of NAG was determined by the fluorescence changes. The assay is highly sensitive to NAG with a low detection limit of 0.75 U L−1 (K = 3) and showed a good linear relationship in the range from 1 to 45 U L−1. This CDs nanoprobe was successfully applied for the determination of NAG activity in human serum and urine samples.