This paper reports the presence of a Kunitz-type protease inhibitor (HGPI) gene in Dolichos biflorus for the first time. A full-length protease inhibitor gene with complete open reading frame of 669 bp encoding 222 amino acids was cloned from D. biflorus using a PCR-based method. BlastN search showed that the HGPI gene shared 100% homology with Cicer arietinum trypsin inhibitor mRNA and 97% with Cajanus cajan protease inhibitor. The deduced amino acid sequence exhibited homology with Kunitz-type trypsin inhibitor from chickpea, pigeon pea and soybean. The deduced amino acid sequence contained N-terminal signal sequence of 18 amino acids and had a molecular weight of 24 kDa. The phylogenetic tree also showed close relationship with Cicer arietinum and Cajanus cajan. Southern blotting revealed the presence of only one copy of HGPI gene in the D. biflorus genome. Homology modeling was employed to predict secondary structure and 3D-structural models for the protease inhibitor. The gene was found to be constitutively expressed in different tissues as determined by semi-quantitative RT-PCR. The novel HGPI gene has been submitted to the GenBank with Accession No. FN666416. The isolated novel HGPI gene will broaden the pool of plant defense genes and might be an ideal choice for developing transgenic crops resistant to insect pests, as well as pathogens. In addition, it could be used as a probe for selection of insect-and pathogen-resistant genotypes.