SARS-CoV-2 virus infection has imposed a significant healthcare burden globally. To contain its spread and decrease infection-related mortality, several vaccines have been deployed worldwide in the past 3 years. We conducted a cross-sectional seroprevalence study to assess the immune response against the virus among blood donors at a tertiary care hospital, Bangkok, Thailand. From December 2021 to March 2022, total of 1,520 participants were enrolled, and their past history of SARS-CoV-2 infection and vaccination was recorded. Two serology test, namely, quantitative IgG spike protein (IgGSP) and qualitative IgG nucleocapsid antibody (IgGNC) were performed. The median age of study participants was 40 years (IQR 30–48) and 833 (54.8%) were men. Vaccine uptake was reported in 1,500 donors (98.7%) and 84 (5.5%) reported the past infection history. IgGNCwas detected in 46/84 donors with the past infection history (54.8%) and in 36 out of the rest 1,436 (2.5%) with no past history. IgGSPpositivity was observed in 1484 donors (97.6%). When compared to unvaccinated donors (n = 20), IgGSPlevel was higher in the donors who had received one vaccine dose (p< 0.001) and these antibody levels increased significantly among those with 3rdand 4thvaccine doses. Factors associated with low IgGSP(lowest quartile) by multivariate analysis included: no past infection history, homologous vaccination, < 3 vaccine doses, and > 90 days duration since last vaccination. In conclusion, vaccine uptake among our study donors was high (98.7%) and IgGSPantibody was observed in nearly all the vaccinated donors (97.6%). Previous SARS-CoV-2 infection, use of heterologous vaccination, vaccines ≥ 3 doses, and duration of the last vaccination >90 days affected IgGSPlevels. Use of serological assays were found beneficial in the evaluation and differentiation of immune response to vaccination, and natural infection including the identification of previous asymptomatic infections.