Background: SARS-CoV-2 infections rapidly spread along with Brazilian territory with heterogeneous transmission and mortality rates, mostly depending on region and period. Investigation of SARS-CoV-2 antibodies is an important tool to understand virus circulation. Given that blood donors are a representative casuistic of a healthy population, the authors evaluated the seroprevalence of IgG and IgM COVID-19 antibodies in 2,806 blood donors from a blood bank located in São Paulo, Brazil. Methods: Aiming to evaluate viral behavior over time, the authors selected samples from blood donors who donated in June and October 2020, and February 2021. To determine whether socio-demographic features affected the seroprevalence, the authors analyzed samples from three different regions from São Paulo (capital, metropolitan and countryside regions) and evaluated predictors as gender, age, educational level, race, and use of public transportation. Results: As expected, the authors observed that seroprevalence increased over time. Seroprevalence was greater in São Paulo city compared to metropolitan and countryside regions, being smallest in the countryside. Characteristics associated with a lower percentage of antibodies were age above 50 years, higher educational level, self-declared Caucasian, and use of individual transportation. Conclusion: In conclusion, blood donors’ samples proved to accurately reflect virus circulation in the healthy population.