Anthocyanin synthase (ANS) catalyzes the conversion of colorless leucoanthocyanins into colored anthocyanins, which is a key enzyme in the anthocyanin biosynthetic pathway. The RsANS gene (GenBank accession number: KR262954) was isolated from‘Xinlimei’radish‘HX12Q-49’in present study. The full length of RsANS was 1,137 bp and included 2 exons and 1 intron. The cDNA length of RsANS was 1,071 bp and encoded a polypeptide of 356 amino acids. Homology analysis showed that RsANS has close relationship with ANS from Chinese cabbage, kohlrabi and mustard. qRT-PCR validation of RsANS revealed that it was expressed at five developmental stages and reached the peak at cortex splitting stage. Furthermore, the expression level of 8 other functional genes and 3 regulatory genes at different developmental stages were investigated. The results showed that the expression pattern of bHLH transcription factor was similar to that of RsANS, CHI, DFR and UFGT, which peaked at cortex splitting stage. The expression pattern of WD40 transcription factor was similar to that of CHS, which gradually increased at seedling, cortex splitting, early expanding and rapid expanding stages, then decreased at mature stage.