Objective The aim of this study was to investigate the incidence of hepatitis C virus (HCV) infection and the risk factors for the infection in maintenance hemodialysis (HD) patients from multiple centers in Jiangsu province. We also measured serum aminotransferase level in these patients. Methods Clinical data of these patients, including demographical information, history of blood transfusion and kidney transplantation, were recorded. Liver biochemistry tests including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Anti-HCV antibody was assayed by enzyme-linked immunosorbent assay (ELISA), and HCV-RNA was detected by real-time polymerase chain reaction (RT-PCR) method. Results (a) Positive anti-HCV antibody was found in 22.1% (176/796) HD patients, and HCV-RNA was found in 80.7% (142/176) patients with positive anti-HCV antibody. (b) Univariate analysis of HCV infected and non-HCV infected patients indicated that the HCV infection was associated with longer duration on hemodialysis (7.13±4.60 vs. 2.73±3.01 year, P＜0.001), blood transfusion (64.2% vs. 45.5%, P＜0.001), dialyzer reuse (51.3% vs. 7.9%, P＜0.001) and kidney transplantation (11.0% vs. 3.5%, P=0.003). Moreover, logistic analysis revealed that longer duration on hemodialysis (OR=1.38, 95% CI=1.24-1.53, P＜0.001) and dialyzer reuse history (OR=10.91, 95% CI=5.52-21.55, P＜0.001) were the independent risk factors for HCV infection. (c) Despite the normal range of average aminotransferase level in HCV infected patients, the average aminotransferase level and the rate of abnormal aminotransferase level were significantly higher in HCV infected patients than in non-HCV infected patients. Conclusions (a) HCV-RNA detection is required in HD patients with positive anti-HCV antibody for the estimation of HCV activity. (b) Serum aminotransferase level may be an insensitive marker for the diagnosis of HCV infection in HD patients, but is useful for the evaluation of hepatitis activity and hepatocytic injury. (c) Comprehensive interventions should be taken to control HCV transmission in HD patients.