In Sub-Saharan Africa, transfusion safety remains a challenge due to the high endemicity of blood-borne infections. This study aimed to determining the seroprevalence of human immunodeficiency virus (HIV), hepatitis B (HBV), hepatitis C (HCV), and Treponema pallidum among blood donors in Ouagadougou. This was a retrospective study in blood donor. HIV 1/2 and HCV antibodies and HBsAg were screened and confirmed with two ELISA (Enzyme Linked ImmunoSorbent Assay). While T. pallidum antibodies were also screened and confirmed with two serology tests. Only samples positive for both tests were counted as positive. Prevalence rates were calculated among first-time blood donors. Of 63,779 registered blood donors, 54,113 (84.84%) were first-time donors. Overall seroprevalences of HIV, HBV, HCV and Treponema pallidum were 2.56%, 11.87%, 5.89% and 3.22% respectively. Seroprevalences of HIV-HBV, HBV-HCV, HBV-T. pallidum and HIV-HBV-HCV co-infections were 0.36; 1.21; 0.54 and 0.02 respectively. The study reports that HIV, HBV, HCV and Treponema pallidum seroprevalences remain high among blood donors. These results highlight a potential infectious risk to blood products recipients.