Chalcone isomerase（CHI）is one of the key enzymes that catalyze the synthesis of isoflavones. In this study,the full-length cDNA of CHI gene was cloned from Astragalus membranaceus,and sequence analysis was carried out. At the same time,the content of isoflavones was determined by ultraviolet spectrophotometry and quantitative analysis was carried out. Lay the molecular foundation. The results showed that the full length of AmCHI gene cDNA is 910 bp,the open reading frame is 660 bp,encoding 219 amino acids,its molecular weight is 24 021.42 Da,the isoelectric point is 5.41,and the Genbank accession number is KY0862871. AmCHI protein is located in the cytoplasm and has no signal peptide. It is a stable hydrophilic protein. Its amino acid sequence is highly similar to legumes. It is presumed to be Type Ⅱ. Total isoflavone content showed root ＜stem ＜leaf,CHI gene expres-sion amount showed root ＜stem ＜ leaf.