The invention discloses a detection kit of gene mutations associated with phenylketonuria. The detection kit comprises ddH2O, 10*buffer (Takara), 2*GC buffer I, dNTP, MgCl2, HSTaq, a multiple PCR mixed primer liquid and an Illumina I5 / I7 primer. The detection kit has a wide detection range, comprehensively detects phenylketonuria caused by five gene defects, sequencing detection is conducted on the upstream 500bp of a genetic transcription start site, 5'UTR, a coding region, 8pp intron sequences of a splice site and transcription terminators, it is predicted that the mutation detection rate can reach 95% or above, and the detection kit can be better applied to clinical practice.