Hepatitis C virus (HCV) infection is a serious and rising global healthcare problem. One critical challenge to tackle this disease is the lack of adequate diagnosis. Here, we develop a multiplex microfluidic paper-based immunoassay, as a novel diagnostic approach, to detect human IgG antibody against HCV (anti-HCV). The paper substrate, highly flammable nitrocellulose (NC), is patterned under ambient temperature by craft punch patterning (CPP) to generate multiple test zones. On the basis of superior merits of patterned paper, this new diagnostic approach demonstrates the key novelty to unprecedentedly combine segmented diagnostic assays into a single multiplex test. The generated diagnostic results are not only informative but can be rapidly and cost-effectively delivered. It would significantly transform the clinical pathway for unwitting individuals with HCV infection. This work highlights the promising role of microfluidic paper-based immunoassays in tackling the diagnostic challenge for the HCV pandemic as well as other diseases.