Giant freshwater prawn (Macrobrachium rosenbergii) is an important cultured species in China. Molting is closely related to the growth and breeding of M. rosenbergii. Chitinase, a member of the glycoside hydrolase family 18, plays vital physiological roles in crustaceans, including molting, digestion, and immunity. The molting cycle of M. rosenbergii consisted of five stages, named postmolt Ⅰ (A), postmolt Ⅱ (B), intermolt (C), premolt (D), and molt (E). To understand the characteristics and molecular dynamics of chitinase in the molting cycle of M. rosenbergii, the chitinase gene of M. rosenbergii (MrChi3B) was cloned. Thereafter, polyclonal antibody against the chitinase was generated in rabbit. The cDNA of MrChi3B spans 1 143 bp that encodes a protein of 380 amino acids (aa) with a predicted molecular weight of 41.91 ku. Phylogenetic analysis revealed that chitinase contains the GH18 family catalytic domain, and it was closely related to chitinase of M. nipponense with 94% homology at the aa level. The expression profile of MrChi3B in six different tissues (eyestalk, gill, stomach, muscles, endocuticle, intestine) and at different stages of the molting of M. rosenbergii was investigated by quantitative real-time PCR (qRT-PCR) and Western blot (WB) analysis. The results showed that the MrChi3B was ubiquitously expressed in all the tested tissues, whereas its expression was significantly increased in stomach, skin, and muscle at A, B stages. At the stage C, highest expression of MrChi3B was observed in the stomach. At the stages A, B, the expression of MrChi3B in the endocuticle and muscle reached the highest. The expression of MrChi3B in the intestine has increased during the stage D and E. Relative lower expression of MrChi3B was observed in the eyestalk at all the stages. The results will shed a new light on the further study of the function of MrChi3B from M. rosenbergii.