To clone and characterize a novel bi-functional alpha-amylase/subtilisin inhibitor (LASI) from the rhizome of Ligusticum chuanxiong, a traditional Chinese medicine. The LASI showed strong homology with members of the Kunitz trypsin inhibitor family. Its putative amino acid sequence has a 40 % identity with that of the alpha-amylase/subtilisin inhibitor from rice. LASI gene without signal peptide was expressed in E. coli Rosetta. After purification, the recombinant LASI protein was inhibitory against not only alpha-amylase from porcine pancreas, Helicoverpa armigera, Spodoptera litura and Plutella xylostella, but also subtilisin A, but not against trypsin or chymotrypsin. In addition, the expression level of LASI in rhizome was higher than that in leaf and LASI expression was enhanced by salt, chilling and drought treatment. This is the first member of the Kunitz-protease inhibitor family identified in traditional Chinese medicine and it might be involved in the plant defense responses against lepidopterous pests, microorganisms and abiotic stresses.