Abstract: Cyprinus carpio L., one of the largest farmed freshwater fish species in the world, can be used as an important food source for humans and has important economic value as ornamental animal. But so far, it is unclear for the molecular regulation network of muscle formation that is an important economic trait of Cyprinus carpio L.. In our early analysis of the Cyprinus carpio L. genome and microRNA expression profiles, we found that miR-1 and miR-133 highly express during Cyprinus carpio L. muscle development. Therefore, we cloned the intergenic enhancer sequence of miR-1-2 and 133a-1 from Danio rerio and Cyprinus carpio L. and conducted in vivo and in vitro studies to determine whether it has muscle specificity. We also investigated whether the transcription factor MyoD regulate this sequence by the luciferase reporting system. The results of in vivo experiments indicated that the intergenic sequences of miR-1-2/133a-1 was muscle-specific in both Danio rerio or Cyprinus carpio L. with the conserved region (cr, containing E-box), and its expression indicated by GFP was significantly higher than other mutants after 72h. In vitro cell experiments showed that the luciferase activity after muscle differentiation was significantly induced in the experimental group containing the cr sequence. Further studies showed that the activity of the intergenic sequence of miR-1-2/133a-1 was regulated by MyoD in both Danio rerio and Cyprinus carpio L.. These results lay the foundation for perfecting the muscle development mechanism of fish and the molecular breeding of fish in the future.