MicroRNAs (miRNAs) are endogenous small non-coding RNAs that participate in diverse biological processes via degrading the target mRNAs or repressing translation. In this study, the regulation of miRNA to the RLR (RIG-I-like receptor) signaling pathway by degrading the target mRNAs was researched in miiuy croaker. MDA5, a microRNA-145–5p (miR-145–5p) putative target gene, was predicted by bioinformatics, and the target sites from the 3′untranslated region of MDA5 transcripts were confirmed using luciferase reporter assays. Pre-miR-145 was more effective in inhibiting MDA5 than miR-145–5p mimic, and the effect was dose- and time-dependent. The expression patterns of miR-145–5p and MDA5 were analyzed in liver and kidney from miiuy croaker. Results implied that miR-145–5p may function via degrading the MDA5 mRNAs, thereby regulating the RLR signaling pathway. Studies on miR-145–5p will enrich knowledge of its functions in immune response regulation in fish, as well as offer a basis for regulatory networks that are composed of numerous miRNAs.