The invention discloses a marking method of litopenaeus vannamei EST microsatellite locus primers. The method comprises the steps of sequence lookup of microsatellite loci, microsatellite primer design, microsatellite locus primer amplification effect and specific detection and microsatellite locus polymorphism detection. Six pairs of EST microsatellite marked specific primers are disclosed. The method is simple, easy and convenient to operate, good in repeatability, stable and reliable. An EST sequence is obtained through transcriptome sequencing, SSR locus information is excavated, the primers are designed, a litopenaeus vannamei specific SSR marker system is constructed, and the method is applied to litopenaeus vannamei family identification, germplasm resource genetic diversity analysis and litopenaeus vannamei molecular genetic breeding or rearing assistance. By adopting the method, markers of different families can be effectively identified, inbreeding and the loss of genetic diversity can be avoided, and the sustainable development of selective breeding of litopenaeus vannamei can be guaranteed.