BACKGROUND Screening of transfusion-transmissible infections (TTIs) among blood donors can be a cost-effective approach to monitor the prevalence, distribution, and trends of the infections among healthy-looking individuals. The study aimed to determine the seroprevalence of four TTIs, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and syphilis, among blood donors in Kathmandu, Nepal. METHODS A total of 21,716 units of blood were tested for the presence of anti-HIV 1/2 IgG/IgM, HBsAg, anti-HCV IgG/IgM, and anti-Treponema pallidum IgG/IgM/IgA using commercial ELISA kits following standard protocols. Statistical analysis was performed using WinPepi Ver 3.8. RESULTS Seroprevalence of HIV, HBV (HBsAg), HCV and syphilis were observed to be 0.12% (95% CI = 0.08-0.18), 0.47% (95% CI = 0.39-0.57), 0.64% (95% CI = 0.54-0.75) and 0.48% (95% CI = 0.40-0.59) respectively. TTIs were dominant among male blood donors compared to female blood donors. Higher HCV seroprevalence among males compared to females was statistically significant. HIV prevalence was highest among blood donors in the age group 31 to 40 years (P > 0.5). HBV, HCV and syphilis prevalence was highest among blood donors 41 to 50 years age group, 21 to 30 years age group, and 51 to 60 years age group respectively (P < 0.05). HIV and HBV prevalence was relatively higher among first-time donors, whereas HCV and syphilis was relatively higher among the repeated donors (P > 0.05). CONCLUSIONS It is of utmost importance to continue screening donated blood with highly sensitive and specific tests and to counsel donors who are positive to any of the above infections. It is absolutely necessary to avoid the transmission of infection from repeat donors.