N-acetyl-beta-D-glucosaminidase (NAG) is an important biomarker for early clinical diagnosis of renal disease, suggesting the necessity to develop a fast and sensitive method for its detection. In this paper, we developed a fluorescent sensor based on polyethylene glycol (400) (PEG-400)-modified and H2O2-assisted etched sulfur quantum dots (SQDs). According to the fluorescence inner filter effect (IFE), the fluorescence of SQDs can be quenched by the p-nitrophenol (PNP) generated by NAG-catalyzed hydrolysis of p-Nitrophenyl-N-acetyl-β-D-glucosaminide (PNP-NAG). We successfully used the SQDs as a nano-fluorescent probe to detect the NAG activity from 0.4 to 7.5 U·L-1, with a detection limit of 0.1 U·L-1. Furthermore, the method is highly selective and was successfully used in the detection of NAG activity in bovine serum samples, suggesting its great application prospect in clinical detection.