Objective To understand the awareness of basic knowledge, diagnosis and prevention knowledgeabout hepatitis C among medical staff in Anhui province, and evaluate the effect of training to explore the feasibility of training mode and training content. Methods The public health and clinical experts for the prevention and control of hepatitis C were invited to lecture through the styles of seminar and interactive discussion. A face-to-face training in the epidemiology, clinical features, and prevention and control of hepatitis C was conducted to the medical staff. A questionnaire survey was conducted to the medical staff before and after training, respectively. Results The correct rate of the basic knowledge of hepatitis C was over 70% before training among the 169 medical staff who participated in the training. And it was increased to above 87% after training among the 121 medical staff. The correct answer rate on the questions such as "the clinical manifestations of acute hepatitis C", "the definition of chronic hepatitis C", "the time of detecting HCV-RNA after infected with hepatitis C", "the laboratory testing basis for confirmed hepatitis C", "the standard prevention principle for hepatitis C", "the measures of preventing hospital infection for hepatitis C", and "the measures should be taken after occupational exposure for hepatitis C", were improved significantly from 78.7%, 85.8%, 33.7%, 74.0%, 53.3%, 71.0% and 23.8% before training to 94.2%, 96.7%, 65.3%, 99.2%, 66.9%, 82.6% and 47.0% after training (all P＜0.05). Conclusion The awareness of diagnosis and prevention knowledge about hepatitis C among medical staff was not well. We should further strengthen the training of medical staff on knowledge in this area.