Immuno-assay is one of diagnostic methods that usually measures biomarkers associated with cancers. However, this method is complex and take a long time to analyze. To overcome these disadvantages, many immuno-sensing chips have been designed and developed. However, these devices still require an external pump or electrical source. In this study, our group fabricated a capillary microfluidic device using glass and adhesive polyethylene terephthalate (PET) film, which were designed by simply patterning and cutting to make the microfluidic capillary channels. Using capillary force alone, glass microfluidic chip can control the speed of fluid-flow and the flow sequence by adjusting the width of the channel and design. In addition, each flow can push out other flow without mixing. The glass-based capillary microfluidic chip (GCMC) can automatically perform immunoassay in regular order without external devices and it provide an electrochemical signal analysis in an average of 2 min. The concentration of the prostate-specific antigen (PSA), a biomarker of prostate cancer, was measured by cyclic voltammetry (CV). In conclusion, GCMC can detect between a range of 100 pg/ml to 1 μg/ml of PSA and provide high selectivity to PSA.