[Objectives]  Serine protease (SP) is an important proteolytic enzymes, in which serine is an active center. In insects, serine protease is involved in several important physiological processes, such as digestion, growth, the innate immune response and tissue reconstruction. A full-length cDNA sequence of serine protease was obtained from Heliothis viriplaca and its deduced amino acid sequence expressed in an E. coli prokaryotic expression system. Enzyme activity was determined after purification and renaturation. [Methods]  Total midgut RNA was isolated from H. viriplaca and one serine protease cDNA sequence was cloned by RT-PCR and RACE to obtain the full-length cDNA sequence. The cDNA sequence was expressed in an E. coli expression system and the expressed fusion protein was denatured, purified and renatured. The activity of the recombinant protein was determined with BTEE as the substrate. [Results]  The serine protease cDNA sequence from H. viriplaca was named HvSP, which was deposited in GenBank with the accession number KT907053. The sequence length was 1 017 bp which included an open reading frame of 886 bp, encoding 295 amino acids. The molecular weight was about 30.8 ku, and the isoelectric point was 8.27. The similarity of the HvSP protein with that of other lepidopteran insects was 46%-92%. The activity of the recombinant protein was 28.7 U/mL at a pH of 8.5 in Tris-HCl buffer solution. The results of quantitative PCR showed that HvSP was expressed at the mRNA level in multiple tissues, especially in the midgut, but not in the salivary gland. [Conclusion]  A new H. viriplaca serine protease cDNA sequence was obtained. The recombinant protein expressed in E. coli was active after denaturation, purification and renaturation. These results establish the basic physiological and biochemical functions of serine protease in H. viriplaca.