Objective: To establish cut-off values of lysosomal storage disease (LSD)-related enzymes by tandem mass spectrometry. Methods: A total of 26 689 newborns and 7 clinically confirmed LSD children underwent screening for LSDs (glycogen storage disease typeⅡ, Fabry disease, mucopolysaccharidosis type Ⅰ, Krabbe disease, Niemann-Pick disease A/B and Gaucher disease). The activities of LSD-related enzymes were detected by tandem mass spectrometry. The 20% of the median enzyme activity of each batch of acid β-glucocerebrosidase, acid sphingomyelinase, β-galactocerebroside, α-L-iduronidase and acid α-glucosidase, and the 30% of the median enzyme activity of α-galactosidase were taken as cut-off values of corresponding enzymes. The genetic diagnosis was performed in neonates whose enzyme activity was lower than 70% of the cut-off value. Results: The enzyme activities of 7 clinically confirmed cases were all lower than the cut-off values. Among 26 689 newborns, 142 cases (0.53%) were suspected positive for LSDs, including 25 cases of β-galactocerebroside deficiency, 1 case of α-L-iduronidase deficiency, 19 cases of α-galactosidase deficiency, and 97 cases of acid α-glucosidase deficiency. Eight infants were genetically diagnosed with LSDs, including 3 cases of glycogen storage disease type Ⅱ, 3 cases of Krabbe disease, and 2 cases of Fabry disease, with a positive predictive value of about 5.6%. Cut-off values ??of the 6 LSD enzyme activities all showed a downward trend from March to August, and an upward trend from September to December. There was a statistically significant difference in LSD enzyme activity among different months (P<0.05).Conclusion: The established cut-off values of LSD-related enzyme activities detected by tandem mass spectrometry can be used for screening LSDs in neonates, and the enzyme activity would be affected by temperature and humidity.