Abstract: The related factors to the formation and growth traits of fish muscle are important topics in aquatic biology and aquaculture research. The expression and regulation of muscle component genes are essential to its tissue function maintenance and trait control, and the identification of cis- regulatory elements in muscle tissue helps explain the genetic basis of muscle formation. Conserved DNA sequences may be found among cis-regulatory elements, whose regulating genes have similar expression patterns. To predict the regulatory elements for zebrafish muscle gene expression, we explored the conservation features for DNA sequences in proximal non-coding regions of muscle highly expressed genes. By analyzing RNA-seq data of multiple zebrafish tissues from public database, we located muscle highly expressed genes as targets and lowly expressed genes as control, respectively. The GO enrichment analysis of these highly expressed genes confirmed their functions associated with muscle development. By discriminative mode of MEME motif discovery tools and using non-coding region of lowly expressed genes as background, we found five target conserved DNA regions, including six DNA motifs of the same sequence, around 300 bp in length, close to gene start sites of five muscle highly expressed genes. Meanwhile, DNA sequences of these five target regions had high pairwise identities (78.62%—84.19%). The results of qPCR confirmed the remarkably higher expression of these five genes in muscle than other tissues. We constructed an eGFP expression reporter plasmid containing the tol2 transposon system. One of the target regions, a 334 bp fragment at upstream of zgc:9242, was cloned into the plasmid at upstream of the eGFP driven by the base promoter. After the plasmid was injected into zebrafish embryos, a greater proportion of muscle specific fluorescence was observed in embryos carrying the target DNA fragment than in the control group (odds ratio=6.487, P=0.000 at 48 hpf), indicating that the 334 bp DNA fragment may enhance muscle gene expression. Using Tomtom motif comparison tools, we also found the candidate binding sites for Myod and other transcription factors within DNA motifs. Our findings suggest that the DNA motif cluster fragments might act as transcriptional regulatory elements to specifically enhance zebrafish muscle gene expression. These results can help us better understanding the genetic basis for fish muscle gene expression and provide a new strategy for predicting tissue specific cis-regulatory elements by bioinformatics.