Abstract: A simple and efficient method for isolating microsatellites was described. The approach involves small-insert genomic library construction, triplex affinity capture, magnetic separation, and PCR screen. The redundancy of grass carp (AG)-enriched library was 7.2%, the enrichment rate was 68.8-fold, and the accuracy rate of PCR screening was 100%. In (AG) repeats we obtained, perfect, imperfect and compound type occupied 66.87%, 17.17% and 15.96% respectively, and uninterrupted repeat sequences longer than 30 bp accounted for 51%. Polymorphism analysis of 34 (AG) repeat sequence loci indicated that 25 repeats of them were polymorphic with 0.50—0.91 PIC. Further analysis revealed that the informativeness of the (AG) repeat sequence loci increased with an increasing number of repeats and uninterrupted repeats longer than 30 bp exhibited abundant polymorphisms. High enrichment rate and informative microsatellites acquisition rate demonstrated the successful application of Triple-Helix-Mediated affinity capture in the isolation of microsatellites in grass carp, and this will build a good foundation for the improvement of breeding lines, genetic diversity detection and genetic map construction in grass carp.