Objective A case-control study was conducted to evaluate the relationship between endothelial nitric oxide synthase (NOS3) gene polymorphism and essential hypertension in the Han, Miao, and Buyi populations in Guizhou China. Methods DNA was collected from the blood samples of 353 essential hypertension patients and 342 healthy controls from Guizhou province of China. Eight polymorphisms of the NOS3 gene were genotyped using the Sequenom MassARRAY platform. For genetic analysis, SPSS 26.0, Haploview, SNPStats, SHEsis, and MDR were utilized. Results All SNPs (rs11771443, rs1808593, rs753482, rs3918186, rs3918188, rs3918227, rs7830, and rs891512) satisfied the Hardy-Weinberg equilibrium test (P > 0.05). The allele and genotype frequencies of rs7830 and rs1808593 in case-control groups demonstrated significant differences (P < 0.05). Compared to the TT genotype of rs1808593, the TG or GG genotype reduced the risk of hypertension in the Miao population (OR = 0.410, 95% CI: 0.218–0.770, P = 0.006). Compared to the GG or GT genotype of rs7830, the TT genotype increased the risk of hypertension in the overall populations (OR = 1.716, 95%CI: 1.139–2.586, P = 0.010). The CATT (rs3918227-rs391818186-rs1808593-rs7830) haplotype was a risk factor for hypertension in the Miao and Han populations (OR = 1.471, 95%CI: 1.010–2.143, P = 0.044 and OR = 1.692, 95%CI: 1.124–2.545, P = 0.011). The CAGG haplotype in the Miao population was a protective factor against hypertension (OR = 0.555, 95%CI: 0.330–0.934, P = 0.025). The rs3918188, rs1808593, and rs7830 in the Miao population showed an interaction effect on hypertension (P < 0.001). The rs11771443, rs3918188, and rs7830 in the Buyi and Han populations showed an interaction effect on hypertension (P = 0.013 and P < 0.001). Conclusion The single nucleotide polymorphisms rs1808593 and rs7830 of NOS3 gene are associated with essential hypertension in Guizhou ethnic populations.