Objective The difference between fluorescence assay (FA) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) in neonatal hyperphenylalaninemia (HPA) screening was analyzed to provide reference for HPA screening. Methods From October 2016 to September 2018,a total of 12529 dried blood specimens of neonatal plantar filter paper were collected.FA and LC-MS / MS were used to detect the phenylalanine (Phe) concentration in dried blood specimens of filter paper,and LC-MS / MS was used to detect the tyrosine (Tyr) concentration.By detecting the dry filter papers made of fresh urine and peripheral blood of the children,the urinary pterin profile and the activity of dihydropteridine reductase of red blood cells were measured,and the gene was sequenced for the second generation,so as to make clear the diagnosis of HPA for the positive results of HPA screening. Results 43 cases of HPA were found positive by the two screening methods, of which 43 cases were detected by FA, 4 cases by LC-MS / MS, and 4 cases by FA. The positive rate of the two screening methods was statistically significant (χ²=32.423, P < 0.05); the false positive rate was 32.72/10 000 (41/12 529) and 1.59/10000 (2 / 12 529) respectively. 43 cases with positive primary screening were diagnosed with Hpa2. 41 cases of suspected HPA detected by the two methods were excluded after 1 year follow-up. Conclusion Both methods were suitable for HPA screening in neonates,LC-MS / MS detection method could reduce the sample retest rate and save the social cost.