Non-O1 Vibrio cholerae, a member of the Vibrio family, could cause gastrointestinal infection of Macrobrachium rosenbergii and result in significant economic losses. However, few studies on microRNA immunity related to non-O1 V. cholerae infection of M. rosenbergii. The aim of this study was to elucidate the mechanism of miRNA in the potential immune response of M. rosenbergii. to non-O1 V. cholerae MSVC-GY01 infection by transcriptome sequencing. Following quality screening, the control group received 10, 616, 712 clean reads, whereas the infected group received 9,727,616. The miRNA sequences in the two samples are extremely consistent and have a length of roughly 23 nt. In all, 871 known miRNAs were discovered, with 279 differentially expressed miRNAs (DEMs). Meanwhile, 62 novel miRNAs were predicted, including 43 DEMs. In order to understand the immune-related biological functions of DEMs, target genes were predicted. Pathway function annotation analysis showed that non-O1 V. cholerae affected the NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, and Toll-like receptor signaling pathway, suggesting that miRNAs in the hepatopancreas play a key role in immune responses to pattern recognition receptors. Twelve DEMs were randomly selected for Quantitative Real-time PCR (qRT-PCR). Overall, the expression trends of qRT-PCR were consistent with the sequencing results. These findings corroborate the immunomodulatory function of miRNA in M. rosenbergii against non-O1 V. cholerae infection and provide guidance for the prevention and treatment of related illnesses.