Transfusion-transmissible diseases (TTDs) are life-threatening for patients requiring blood transfusion. The Human hepatitis C Virus (HCV), Hepatitis B Virus (HBV), immunodeficiency (HIV) and Syphilis are the most important agents causing transfusion transmissible infection. Therefore, due to the lack of data regarding TTDs prevalence in the last 5 years this study sought to investigate the prevalence of TTDs in Jordan among blood donors in the period between (2020–2023). A retrospective review of the records of healthy donors was carried out from January 1, 2020, to December 31, 2023. The data were collected from Central Blood Bank in Zarqa and 5 main governmental hospitals in the Middle of Jordan. Screening for HBsAg, HBcAb Ab, HCV Ab, HIV I/II Ab and Syphilis using ELISA technique and CMIA on Abbott ARCHITECT i2000 SR analyzer. Based on the records the total number of donors for the region was 67,081 divided on the 4 years’ period as following; 15,754, 16,494, 17,493, and 17,340, respectively. The 4-year average prevalence rate was 0.37% for HBsAg, 3.25% for anti-HBcAb, 0.14% for anti-HCV, 0.005% for anti-HIV I/II and 0.01% for anti-TP. The prevalence rates among centers in the Middle region was approximately equal and the differences in the prevalence rates among years and centers was not statistically significant (P > 0.5).