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Objective To investigate missed detection in the screening for hepatitis C virus (HCV) infection in a general hospital in 2015-2018. Methods A retrospective analysis was performed for the data of 3659 patients who attended People’s Hospital, Peking University, from 2015 to 2018 and underwent the detection of anti-HCV and HCV RNA. Architect I2000 by Abbott and Vitros 3600 by Johnson and Johnson were used to measure anti-HCV, and the Roche Cobas AmpliPrep/Cobas Taqman 96 automatic virus quantification system was used to measure HCV RNA. The specimens were collected from the patients with positive HCV RNA and negative anti-HCV, and Sanger sequencing was used to determine HCV genotype. The patients were followed up to observe the status of HCV infection and clinical conditions. The signal-to-cut-off (S/CO) ratio was used to express the results of anti-HCV detection. GraphPad Prism 5.0 was used to plot the distribution map of the S/CO ratio of anti-HCV. Results Of all 3659 patients, 6 (0.16%) had negative anti-HCV based on at least one reagent and positive HCV RNA, with a mean level of (6.40±1.86)log10 IU/ml. Among these 6 patients, 5 (83%) had acute leukemia and 1 had respiratory disease; among these patients, 1 had good prognosis, 3 had poor prognosis, and 2 died. Conclusion When antibody is used as the primary screening method for HCV infection, the rate of missed detection reaches 0.16%, and most of these patients have poor prognosis. HCV RNA detection should be performed for patients with immunodeficiency to avoid missed detection.