Abstract: Hong Kong oyster (Magallana hongkongensis) is an important economic shellfish cultivated in the coastal areas of southern China. However, breeding diseases frequently happen during cultivation, and they have seriously restricted the sustainable development of Hong Kong oyster farming industry. As an important regulatory factor, microRNA (miRNA) participates in various biological processes of marine species through posttranscriptional regulation of their target genes. In this study, we used high-throughput sequencing technology to create miRNA libraries from gill of Hong Kong oyster under different induction time points of Vibrio parahaemolyticus infection and then explored the miRNAs related to immune regulation via bioinformatics analysis. In total, we obtained 453 conserved miRNAs and 222 novel miRNAs. Subsequently, the unique miRNAs among blank control group/12 h pathogen-injection group, blank control group/24 h pathogen-injection group, 12 h pathogen-injection group/24 h pathogen-injection group were further screened. As result, a number of differentially expressed or highly expressed miRNAs were found in different comparison groups. Among them, miR-10, miR-23, miR-92, miR-100, miR-125, miR-144, miR-145, miR-146, miR-335 and miR-750 have been verified to involve in the immune regulation processes in a variety of marine animals. KEGG enrichment analyses of target genes of differentially expressed miRNAs showed that the target genes of miR-411-5p, miR-4454-5p, novel-m0159-3p and novel-m0187-3p participated in immune-related pathways. These results indicated that the above-mentioned miRNAs may play an important role in the immune regulation of Hong Kong oysters, Thus, it is necessary to further explore on them.