AbstractHepatitis C remains a global health problem, especially in poverty‐stricken areas. A rapid and sensitive point‐of‐care (POC) diagnostic tool is critical for the early detection and timely treatment of hepatitis C virus (HCV) infection. Here, for the first time, we reported a novel molecular diagnostic assay, termed reverse transcription multiple cross displacement amplification integrated with a gold‐nanoparticle‐based lateral flow biosensor (RT‐MCDA‐AuNPs‐LFB), which was developed for rapid, sensitive, specific, and visual identification of HCV. HCV‐RT‐MCDA induced rapid isothermal amplification through a specific primer set targeting the 5′untranslated region gene from the major HCV genotypes 1b, 2a, 3b, 6a, and 3a that are prevalent in China. The optimal reaction temperature and time for RT‐MCDA‐AuNPs‐LFB were 68°C and 25 min, respectively. The limit of detection of the assay was 10 copies per test, and the specificity was 100% for the experimental strains. The whole detection procedure, including crude nucleic acid isolation (~5 min), RT‐MCDA (68°C, 25 min), and visual AuNPs‐LFB result confirmation (less than 2 min), was performed within 35 min. The preliminary results indicated that the HCV‐RT‐MCDA‐AuNPs‐LFB assay could be a valuable tool for sensitive, specific, visual, cost‐saving, and rapid detection of HCV and has potential as a POC diagnostic platform for field screening and early clinical detection of HCV infection.