The invention belongs to the technical field of germplasm innovation of aquatic animals and particularly relates to a method for for breeding litopenaeus vannamei with WSSV (white spot syndrome virus) resistance on the basis of multi-marker combination assistance and an application of the multi-combination markers. SNP marker typing is performed on SNP loci of immune signaling pathways of the litopenaeus vannamei in sensitive groups and resistance groups with a high throughput typing technology, and 6 SNP molecular markers in significant correlation with WSSV resistance are obtained. The optimal resistance maker combination is obtained through screening with a multi-marker interaction analysis method and comprises nLvALF2g-89-A>G, Unigene34569g-97-T>C and TRAF6g-139-A>G, and four genotype combinations including AA-TT-AA, AA-CT-AA, AA-CT-AG and AG-CT-AA of the three markers show an obvious correlation advantage with the WSSV resistance. Candidate markers are provided for disease-resistant molecular breeding of prawns, one novel path is provided for molecular marker assistant resistance breeding of the prawns, and broad application prospect is realized in research of WSSV-resistance variety breeding of the prawns.