Hepatitis C virus (HCV) is silently becoming a major public health problem. Currently, no validated HCV knowledge measures exist. This study aimed to develop and validate a brief measure to assess general knowledge about HCV risk factors, modes of transmissions, and treatment options. A total of 406 individuals participated in this cross-sectional study. All participants completed the proposed 19-item Brief HCV Knowledge Scale. Participants were: HCV mono-infected patients (n = 83), HCV-human immunodeficiency virus (HIV) co-infected patients (n = 24), HIV mono-infected patients (n = 128) community healthcare workers (n = 89), and college students (n = 82). Two-week test-retest data were collected for the college student sample. Psychometric evaluation of the proposed scale demonstrated high levels of validity (content and construct validity) and reliability (internal consistency and retest stability). Factor analysis indicated a one-factor solution, which accounted for 49% of the variance. HCV knowledge was positively correlated with length of time since HCV diagnosis (r = 0.29, p < 0.05). HCV treatment-experienced patients obtained significantly higher HCV knowledge scores (82% correct) than HCV treatment-naïve patients (72% correct) (p < 0.05). HCV knowledge in College students (43% correct) and HIV patients (54% correct) was significantly lower than in HCV patients (77% correct) and community healthcare workers (80% correct) (p < 0.001). Community workers' HCV knowledge was positively correlated with years of HCV work experience (r = 0.30, p < 0.01). This self-administered Brief HCV Knowledge scale has high levels of validity and reliability across patient, healthcare provider and college student populations. It has valuable applications as a clinical teaching tool with patients and healthcare providers and could be used as an outcome indicator in novel HCV educational intervention studies.