CYP83A1 is a key gene in sulforaphane biosynthesis and metabolism. The full length of BoCYP83A1 was obtained from broccoli CDBY10 by the methods of RACE and RTPCR. BoCYP83A1 was 1 509 bp in length, encoded a deduced protein of 502 amino acids, and contained the conserved P450 domain. The expression level of BoCYP83A1 in different cultivars, tissues, or seedlings treated with different hormones was analyzed by realtime fluorescence quantitative PCR. Phylogenetic tree analysis showed that BoCYP83A1 had the closest relationship with Brassica oleracea var. oleracea BoCYP83A1. Tissuespecific expression of BoCYP83A1 in cultivars was different: expression difference in CDBY10 was small. It showed stem>leaf>root in CDBY12，while root > stem > leaf in CDBY14. The treatments of MeJA and SA led to the changes of BoCYP83A1 expression：after the MeJA treatment，the expression of BoCYP83A1 was raised to 1.9 times of the control, and then decreased a little; After the SA treatment，the expression of BoCYP83A1 reduced quickly，and upto 0.1 time of the control at 6 h, then restored gradually to the expression level of control. The results indicated that the expression characteristics of BoCYP83A1 in broccoli cultivars were different, and BoCYP83A1 expression can be regulated by MeJA and SA. Cloning and characterization of BoCYP83A1 gene provide the theoretical basis for breeding new broccoli cultivars with high sulforaphane content.