Serine proteases are enzymes that have serine, histidine and aspartic acid in their active domain and same catalytic mechanisms, which play important roles in many diseases and intracellular signal transduction. In this study, we amplified the coding sequence and promoter sequence (2605 bp) of serine protease I-1 from Japanese flounder Paralichthys olivaceus (PoSP I-1). We identified 9 single nucleotide polymorphisms (SNPs) and 28 SNPs in the coding sequence and promoter sequence of PoSP I-1, respectively. The analysis of all SNPs indicated that the frequency of SNP365A/G in the coding sequence of PoSP I-1 is significantly different between the resistant group (60%) and the susceptible group (40%) (P=0.01). Vibrio anguillarum injection reduced the expression of PoSP I-1 in the susceptible individuals but induced its expression in the resistant individuals. Moreover, PoSP I-1 in the resistant individuals is higher than that in the susceptible individuals. These results implied that PoSP I-1 is involved in the resistance of Japanese flounder to V. anguillarum. 365A/G of PoSP I-1 is a potential marker associated with the resistant Japanese flounders. In conclusion, PoSP I-1 365A/G is a candidate marker that could apply to disease resistant selective-breeding of Japanese flounder in the future.