Hepatitis C virus (HCV)-RNA amplification is a costly procedure in terms of time and reagents. Consequently, the search for more a cost-effective specific HCV diagnostic method is of great interest. Capillary zone electrophoresis (CZE) methods that detect HCV in serum, plasma, whole blood, and ascites without the need for sample pretreatment are not currently available. Here, a CZE method was developed that detects a larger specific peak in serum and other body fluids of HCV-infected patients than that found in healthy or hepatitis B virus (HBV)-infected individuals. The nature of the HCV peak was investigated using biochemical treatments, including RNase, DNase, and chymotrypsin enzymes. Electroeluted HCV peak was applied to transmission electron microscopy; electron micrographs showed that the HCV peak was attributed to virus-like particles with diameter and morphological properties similar to non-enveloped HCV nucleocapsids. The determination of CZE-HCV and HCV-RNA levels using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) in 258 subjects revealed that these two tests were highly correlated (r = 0.92, p < 0.0001). One important issue of HCV testing is the storage conditions of serum to obtain reliable results. Serum samples at -20 °C showed the best preservation of the HCV peak up to one year. In conclusion, we detected HCV using CZE in a microliters volume from different body fluids. Besides the stability of samples in maintaining their peak height, the HCV-CZE test is rapid (<15 min) and a well-suited and low-cost technique. Thus, a major improvement in the quantitative diagnosis of HCV infection was established.