Pseudomonas aeruginosa exoS gene contains important replacement (non-synonymous) single-nucleotide polymorphism (SNP) loci, of which mutations in loci 162 (G162A) and 434 (G434C) in exoS greatly affects virulence. The present study aimed to develop an SNP-based classification method for exoS loci (G162A and G434C), using magnetic enrichment polymerase chain reaction, magnetic separation, and dual-color fluorescence to provide a technical basis for understanding the T3SS genotypic variation. The two SNP loci in 3 P. aeruginosa standard strains, ATCC27853, ATCC9027, and CMCC10104, were analyzed using this method. The two SNP loci of all these strains were found to be of the wild-type subtype. G values were greater than 0.8 and I values were greater than 3; hence, the classification yielded statistically significant results. In addition, G162A and G434C SNP loci in 21 clinical isolates were analyzed using this method for monitoring clinical mutations. In the G162A and G434C SNP loci, 57.1% and 80.9% of isolates were of the wild-type subtype; 23.8% and 14.3%, mutation subtype; 9.5% and 4.8%, heterozygous subtype, respectively. In a word, SNP genotyping of loci G162A and G434C in exoS was established using magnetic separation and dual-color fluorescence hybridization, and the method was optimized.