Introduction: Blood transfusion is a life–saving measure in emergencies and is important for the medical treatment of every patient. Among all adverse effects of transfusion, transfusion transmitted infections (TTI) are very important. Although the risk of transfusion–transmitted infections today is lower than ever, the supply of safe blood products remains subject to contamination with known and yet to be identified human pathogens. Our aim was to study the seroprevalence of leptospirosis among voluntary blood donors in Chennai. Materials and Methods: A total of 100 voluntary healthy blood donors selected from different regions of Chennai were included in the study. Five ml of blood was collected directly from the donor in a sterile plain test tube. Serum was stored at 40 C and –200 C in two separate aliquots for MAT and PCR studies respectively. Another 5 ml of blood was collected from donor’s blood bag immediately after collection and used within 4 hours for DFM study. Results: Prevalence of leptospirosis among voluntary blood donors in our study was found to be 21%. Of the 100 samples studied, 31 were positive by DFM, 40 by MAT, 21 by both DFM and MAT; however, none of the samples were found positive by PCR. Conclusion: Leptospira survived for 48 hrs in stored plasma at 4–6°C and 24 hrs in fresh frozen plasma stored at –18oC. DFM technique can be used as a simple, rapid and inexpensive means of leptospira detection. Leptospiral screening can be made mandatory in endemic regions like Chennai after undertaking a larger study.