Phenylketonuria (PKU), caused by phenylalanine hydroxylase (PAH) gene variants, is a common autosomal inherited metabolic disease. So far, 1111 PAH variants have been revealed. The residual activity of the PAH variants is the key determinant of the metabolic phenotype and BH₄ responsiveness in PKU patients. In this study, the spectrum of PAH variants in 1083 Chinese PKU patients was analyzed. Then 20 variants (p.L52F, p.R86P, p.L128P, p.L142P, p.D163N, p.C203G, p.E214G, p.F260L, p.M276T, p.L311R, p.P314A, p.L364F, p.Q375H, p.F382I, p.A395S, p.V412D, p.E108*, p.C203*, p.C284* and p.E353*) were expressed in COS-7 cells. The residual activities and protein expression levels were detected by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and Western blotting, respectively. We compared the results of the phenotypic prediction based on APV and PAH activity respectively, and further explored the relationship between residual activity and phenotype in PKU patients. We reported 9 newly discovered PAH variants for the first time, thereby expanding the spectrum of PAH variants. Among the 20 variants in our assay, 8 variants showed mild impaired residual activities (48-92%) and approximately normal protein expression levels compared to the wild-type PAH. In contrast, 9 variants showed severely impaired residual activities (0-34%) and reduced protein expression. However, three variants (p.L52F, p.F260L and p.P314A) showed impaired residual activities (5%, 32% and 29%), although the proteins were well expressed. We assigned APV scores for 14 variants, in which the results of the phenotypic prediction were consistent for 12/14 (86%) variants based on APV and residual activity respectively, and the residual activity correctly predicted 17/22 (77%) of the patients. Our study helped to further understand the genotype-phenotype correlation in PKU patients.