In order to explore the role of miR-137 in the color formation of koi, the precursor sequences of miR-137 in 14 fish species were aligned and constructed phylogenetic tree by maximum likelihood (ML). Besides, The expression levels of miR-137 in different developmental stages and tissues also detected. Then, the target genes of miR-137 was predicted and analyzed by GO and KEGG analysis. In addtion, the luciferase reporter was used to verify the availability of miR-137 with its targets, and the expression levels of miR-137 and target genes in different development stages and tissues were checked and the correlation analysis between them was also conducted. The results showed that the precursor sequence similarity of miR-137 between koi and carp was the highest, and miR-137 sequences exhibited high conservation among all fish species. A great number of predicted target genes enriched in melanogenesis, pigment cell differentiation, pteridine-containing compound metabolic process and so on. Real time PCR results showed the expression of miR-137-3p climbed the peak on 11 dph and rapidly dicreased in the following stage. miR-137 was expressed in kinds of tissues, especially significant in eye and muscle. Besides, the skin and fins, the pigment cell enriched tissues, also had a higher expression and the expression of miR-137 in white tissues was higher than the red. The luciferase reporter analysis certified that miR-137 bound with the mitfa 3＇-UTR to inhibit its expression but did not appeared significant inhibitory effect on sprb, and miR-137 was negatively correlated with the expression of mitfa and sprb in different developmental stages, but not in tissues. All the above results confirmed miR-137 was highly conserved during fish species evolution and displayed correlation with the koi color formation, in which miR-137 might regulate mitfa involving in the body color formation. This study provides basic research data for further exploring the role of miR-137 in koi color formation.