Chitinase is a multi-gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase-4 gene (PmChi-4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi-4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi-4 protein was 67.7 kDa and shared 61%–88% identity with the type of Chi-4s from other crustaceans. Quantitative real-time (qRT-PCR) analysis indicated that PmChi-4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi-4 was expressed throughout the whole larvae stages, and the highest level of PmChi-4 transcripts was detected at Mysis3 stage, which indicated that PmChi-4 may be involved in larval metamorphosis. In order to know whether PmChi-4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi-4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi-4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi-4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon.