Objective To isolate microsatellite DNA markers from genome of Mugilogobius chulae for further identification of its genetic background. Methods A microsatellite-enriched genomic library for Mugilogobius chulae was constructed by magnetic-bead enrichment method using biotinylated (AC)₁₅ probe. After a series of steps, the obtained microsatellite markers were assessed and evaluated versus that of wild population of Mugilogobius chulae. Results 74 repeated sequences containing microsatellite sequences were obtained from 95 randomly picked positive clones. 33 primers were designed and synthesized. All of these loci were selected for polymorphism analysis in a wild population of Mugilogobius chulae, and 12 of them showed polymorphism. The mean number of alleles (Na), the number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC) were 2.9167, 2.2311, 0.4583, 0.5633 and 0.4474, respectively. Conclusions The microsatellites developed in this study represent the first nuclear markers described in Mugilogobius chulae and will be useful tools for genetic studies of Mugilogobius chulae.