Objective To explore the clinical significance of combined detection of thyroid stimulating hormone (TSH) and free thyroxin (FT4) in dried blood spots in screening for congenital hypothyroidism (CH) in neonates. Methods The TSH and FT4 levels in dried blood spot were measured by time-resolved fluorescence immunity in live born neonates from June to December 2013. If the screening was positive, the blood was drawn and the serum TSH and FT4 were measured and compared with the results from dried blood spots. Results In a total of 31 199 neonates screened, 12 cases were diagnosed with CH and the prevalence rate of CH was 1/2 600; 4 cases were hyperthyropinemia and no pituitary CH was detected. There was no significant difference between TSH or FT4 levels in dried blood spot and those in serum in neonates diagnosed with CH (P>0.05). Conclusions Combined detection of TSH and FT4 in dried blood spot can be used for neonatal screening of CH. It can be applied for early distinguishing CH from hyperthyropinemia, and also helpful for early diagnosis and treatment of central CH.