IiCYP79F1 gene was cloned from Isatis indigotica Fort. by RTPCR method, and its bioinformatics and expression analysis were performed. The results showed as follows: (1) the full length of gDNA for IiCYP79F1 was 2 109 bp, including two introns and three extrons; and the full length of ORF was 1 626 bp, encoding 541 amino acids (GenBank accession Number: KY774689.1). Bioinformatics analysis indicated that the secondary structure of IiCYP79F1 protein was mainly composed of αhelix (43.81%), random coil (35.49%), extended strand (13.68%) and βturn (7.02%); and its amino acid sequences had a high degree of identity with that of CYP79F1 in Brassica oleacea, Brassica napus, Brassica rapa and Eruca sativa. The phylogenetic analysis exhibited that IiCYP79F1 protein of I. indigotica Fort. had the closest relationship with Eruca sativa. (2) qRTPCR analysis demonstrated that IiCYP79F1 gene was expressed in spatiotemporal specificity and was highly expressed in stem and the seedling stage. MeJA, Ag⁺, glucose and mechanical injury promoted the expression of IiCYP79F1 gene, while SA and low temperature treatments had inhibiting actions. The results could lay the foundation for the further exploration of the function of IiCYP79F1 on the synthesis of glucosinolates, and provide a new idea for the new germplasm resources development.