Objective To explore the feasibility of detecting iduronate 2-sulfatase (IDS) activity in dried blood spot (DBS) for screening of mucopolysaccharidosis type Ⅱ (MPS Ⅱ) in newborns. Methods The IDS activity of DBS was detected by fluorescence quantification method, and MPS Ⅱ screening was performed on 2202 male newborns born from February to April 2021 in the Neonatal Disease Screening Center of the hospital. The 51 confirmed children with MPS Ⅱ, 15 female carriers of MPS Ⅱ and 32 non-MPS Ⅱ children (the control group, all male) collected by the Department of Pediatric Endocrinology and Genetic Metabolism from March 2012 to December 2020 were taken as the pre-screened population. A preliminary screening cut-off value for positive cases has been established, and those who test positive will be recalled for retesting of enzyme activity and urine mucopolysaccharides and IDS gene testing. Results The median age of 51 children with MPS Ⅱ was 4.2 years, the median age of 15 female carriers with MPS Ⅱ was 27.5 years, and the median age of 32 control patients was 4.5 years. ROC curve was drawn, and the optimal threshold of DBS for detection of blood IDS activity was 9.59 ρmol/(punch‧20h) in 98 pre-screened patients, with sensitivity of 100%, specificity of 22.7% and AUC of 0.962 (0.928-0.995). The consistency analysis between the white blood cell method and DBS method showed that Kappa=0.96 (P<0.001). The activity level of IDS in 2202 neonates was detected by DBS method. The median value of IDS activity was 58.22 ρmol/(punch‧20h) and 20% of the median value was 11.64 ρmol/(punch‧20h). Whether the DBS IDS activity ≤9.59 ρmol/(punch‧20h) or≤11.64 ρmol/(punch‧20h) was taken as the positive cut-off value, 6 cases (0.27%) were detected as positive and recalled. Among the initial screening positive cases, although 3 patients had a qualitatively positive urine mucopolysaccharides, the electrophoresis showed only chondroitin sulfate band without dermatan sulfate and heparan sulfate bands. Genetic testing found five cases carrying the mother- derived IDS missense variant of c.851C>T (p.P284L) and one case carrying c.1499C>T (p.T500I), all of which were classified as benign variants. Conclusions The IDS activity test on DBS can effectively and promptly identify suspected patients with MPS Ⅱ and can be used for newborn screening of MPS Ⅱ. However, a large sample size study is still needed to determine the accurate positive cut-off value.