Toll-like receptors (TLRs) play a crucial role in the recognition of immune reactions against invading pathogens. The molecular regulation mechanisms of TLR expression in aquatic organisms remain unclear. MicroRNAs (miRNAs) are small non-coding RNAs that are critical adjustors of immune signaling pathway at the post-transcriptional level and play critical roles in intricate networks of host–pathogen interactions and innate immunity. The critical role of TLRs in host defense for discerning certain kinds of pathogen associated molecular patternsand striking a cascade immune response in fish have been demonstrated. Miiuy croaker TLR13 significantly increased after infection with Vibrio anguillarum, which suggests that mmiTLR13 plays an important role in innate immunity. In this study, the role of miR-8159 was explored in regulating TLR13, which is involved in inflammatory responses in miiuy croakers. Bioinformatics was used to predict miR-8159, which has a direct negative regulatory effect on TLR13 in miiuy croaker. Afterward, the dual luciferase reporter assay containing miRNA mimics or inhibitors and pre-miR-8159 showed that miR-8159 was the direct negative regulator of TLR13 in miiuy croaker. Moreover, miR-8159 downregulated the expression of TLR13 in the transcription level. The expression of miR-8159 could be upregulated by V. anguillarum challenged miiuy croaker and LPS exposure macrophages. Thus, miR-8159 could be induced by V. anguillarum and may function as a negative regulator of TLR13 in the immune response of miiuy croakers.