Background: Human leukocyte antigen-G (HLA-G)and Toll-like receptor-9 (TLR-9)play a role in the regulation of autoimmune diseases and inflammatory processes. Aim of the study: To detect the HLA-G + 3142G > C gene polymorphism that associated with the susceptibility to SLE patients and associated with Hepatitis B infection and TLR-9 serum level. Patients and methods: This study was done on 75 SLE patients and 75 healthy control groups. Genotyping of HLA-G + 3142G > C were detected by PCR and PCR-RFLP methods. In addition to the estimation of Hepatitis B surface (HBs)antigen status by immunochromatography technique and TLR-9 serum level by ELISA technique. Results: The HLA-G + 3142G > C gene polymorphism between the SLE patients and controls in CC, CG and GG genotyping (5.33% vs. 45.34%; OR = 0.07; P = 1.0 × 10−8, 21.33% vs. 37.34%; OR = 0.46; P = 0.048, 73.33% vs. 17.33%, OR = 47.45; P = 4.5 × 10−12 respectively). In addition, there was a significant increase frequency of G allele in SLE patients compared to controls (0.84 vs. 0.36). While the C allele showed a significantly decreased frequency in patients compared to controls (0.16 vs. 0.64). The seropositive status of HBs antigen showed no significant difference between the SLE patients' group and controls (OR = 7.3, 95%CI = 0.38–140.81). While the mean of TLR-9 serum level was significantly increased in SLE patients' group compared to the control group (399.9 ± 66.7 pg/ml vs. 122.2 ± 28.5 pg/ml; p ≤ 0.05). Conclusion: The HLA-G + 3142G > C homozygous genotype GG and G allele were more associated with SLE patients. The HBs antigen showed no significant association with SLE patients. While, TLR-9 serum level showed a significant association with SLE development.