Background and Aim: Trypanosoma evansi infection has been reported in Thai livestock such as beef and dairy cattle. However, there is little information on T. evansi infection in bullfighting cattle in Southern Thailand. The aim of this study was to investigate the infection of T. evansi in bullfighting cattle presented for health checks at the Animal Hospital, Faculty of Veterinary Science, Prince of Songkla University, Thailand. Materials and Methods: Blood and serum samples were collected from 177 bullfighting cattle from April 2016 to February 2017 after bullfighting matches. Animal inspected showed signs of fever, weight loss, or exercise intolerance. Investigation of T. evansi infection was tested using polymerase chain reaction (PCR) with TBR primers and using indirect enzyme-linked immunosorbent assay with T. evansi crude antigen. Results: The seroprevalence of T. evansi in bullfighting cattle was 22.60% (40/177). The PCR results detected no parasite DNA in this study. However, bullfighting cattle may serve as T. evansi reservoirs. Conclusion: Health checking procedures for T. evansi should be promoted for bullfighting events so that infected animals can be quarantined in the preparatory stages of such events.