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Background: The safety of blood essential component of blood screening program. Different countries have different health standards. Maintaining effective blood screening is critical for blood recipients. However, the microbial agents that are transmissible from blood to blood are particularly hazardous for blood transfusion services because such microbial agents cause morbidity and mortality in the blood recipients. Previously conducted studies have just focused on seropositivity rate of TTIs, the aim of present study to study seropositivity and association with demographic factors. Methodology: A cross-sectional study was carried out in the Hematology Section of Diagnostic laboratory of Isra University, Hyderabad in April 2019 to May 2019. Basic demographic data was collected from participants through structured questionnaire. The seropositivity was assessed using ICT and Elisa method. Results: A total 353 blood donors participated in study. The mean age of blood donors was 27.59±5.88 years. The seropositivity of HBV among the blood donors of Hyderabad was 1.1%, HCV was 5.1%, HIV was 0.8%, syphilis was 2.3%. Among risk factors a significant association was noted between HCV and repeated blood donation [χ2=4.05 p=0.04]. Conclusion: There is dire need to launch an effective health education program to aware people about the prevention of infectious diseases.