End-stage renal disease patients who receive long-term hemodialysis have a higher incidence and prevalence of hepatitis C virus infection than the general population. It has been recommended that hemodialysis patients should check for hepatitis C virus antibodies (HCV-Ab) every 6 to 12 months to detect suspected cases. This article described an incident in 2020 with three long-term hemodialysis patients in Southern Taiwan who were found to have HCV-Ab seroconversion within one year, leading to a presumed hospital outbreak. Health authorities and the hospital investigated the scale of this outbreak and transmission route, and implemented infection control measures. The hospital immediately convened an interdisciplinary team to assess the infection status of the patients and determine the follow-up measures. A strategy of managing hemodialysis patients with HCV-Ab seroconversion was also established. In the end, hepatitis C virus nucleic acid tests of the three patients were all negative. This experience can provide a reference for the personnel in hemodialysis units to manage similar events.