Background The prevalence of transfusion transmitted infections (TTIs) among blood donors varies across different geographical populations. Establishing the sero-prevalence of the disease among blood donors is important to informing the direction of preventive and control strategies. Objective The aim of this study was to determine the sero-prevalence of transfusion transmitted infections among voluntarily blood donors at Dessie Blood Bank, North East Ethiopia. Methods and Materials A cross-sectional study was conducted from November 10 to December 12, 2018. A total of 384 blood donors were conveniently included in this study. Socio-demographic data and other factors were collected using a pre-tested structured questionnaire. Five milliliters of venous blood was collected using a sterile test tube from each blood donor and the blood was allowed to clot; then, serum was separated by centrifugation for laboratory investigation. Serum samples from blood donors were tested by enzyme-linked immunosorbent assays (ELISA) for the presence of hepatits B surface antigen (HBsAg), and antibodies to human immunodeficiency virus (HIV-1/2), hepatits C virus (HCV), and Treponema pallidum . Logistic regression was used to explore risk factors associated with each transfusion transmissible infection. Results From a total of 384 blood donors, 24 (6.25%) of them had serological evidence for at least one infection. The overall sero-prevalence rates of HBV, HCV, HIV, and syphilis among blood donors were 4.2%, 0%, 0.26%, and 1.82%, respectively. Educational status was significantly associated with HBV infection. Multiple sexual behaviors had statistically significant association with syphilis. Conclusion In conclusion, 6.25% of blood donors still harbor transfusion transmissible infections despite recent blood donation safety improvements with the greater majority (4.2%) of cases caused by HBV infection.