Parvalbumins (PVALBs) are particularly abundant in the fast-contracting muscles and correlate positively with muscle relaxation speed in amphibians and fishes. MiRNAs play important roles in diverse biologic processes via binding to the 3' untranslated region (3'UTR) of the target mRNAs. In the study, four PVALB isoforms, named as PVALB1, 2, 3, and 4, were identified in the mandarin fish (Siniperca chuatsi) fast muscle and PVALB4 exhibited the highest expression level among them. By bioinformatics analysis, a putative miR-181a binding site in PVALB4 was detected and the direct interaction between miR-181a and PVALB4 was confirmed with the luciferase reporter assay. Further, when miR-181a was inhibited, it substantially increased PVALB4 mRNA expression level and the muscle relaxation rate in vivo. Taken together, the obtained results suggest that miRNA-181a/PVALB4 is an evolutionarily conserved miRNA-target pair and their interaction is correlated with muscle relaxation rate in the mandarin fish. Therefore, the study revealed a novel molecular mechanism in the regulation of skeletal muscle relaxation in fish.