CYP83A1 and CYP83B1 are two key synthesis genes in the glucosinolate biosynthesis pathway. CYP83A1 mainly metabolizes the aliphatic oximes to form aliphatic glucosinolate and CYP83B1 mostly catalyzes aromatic oximes to synthesis corresponding substrates for aromatic and indolic glucosinolates. In this study, two CYP83A1 genes named BcCYP83A1-1 (JQ289997), BcCYP83A1-2 (JQ289996) respectively and one CYP83B1 (BcCYP83B1, HM347235) gene were cloned from the leaves of pak choi (Brassica rapa L. ssp. chinensis var. communis (N. Tsen & S.H. Lee) Hanelt) "Hangzhou You Dong Er" cultivar. Their ORFs were 1506, 1509 and 1500 bp in length, encoding 501, 502 and 499 amino acids, respectively. The predicted amino acid sequences of CYP83A1-1, CYP83A1-2 and CYP83B1 shared high sequence identity of 87.65, 86.48 and 95.59% to the corresponding ones in Arabidopsis, and 98.80, 98.61 and 98.80% to the corresponding ones in Brassica pekinensis (Chinese cabbage), respectively. Quantitative real-time PCR analysis indicated that both CYP83A1 and CYP83B1 expressed in roots, leaves and petioles of pak choi, while the transcript abundances of CYP83A1 were higher in leaves than in petioles and roots, whereas CYP83B1 showed higher abundances in roots. The expression levels of glucosinolate biosynthetic genes were consistent with the glucosinolate profile accumulation in shoots of seven cultivars and three organs. The isolation and characterization of the glucosinolate synthesis genes in pak choi would promote the way for further development of agronomic traits via genetic engineering.