In this study, we carefully designed microfluidic devices, and the surface of a detection chamber was coated with gold for the direct assembly of proteins. A microfluidic-based immunosensor to detect human H1N1 influenza was developed into a low-cost immunosensor based on the exploration of fluorescence signals. The detection of a specific antibody among serological assays in blood samples was performed in the microfluidic biosensor chip by immunoreactions between the GBP-recombinant influenza hemagglutinin antigen (GBP-H1a) fusion protein and its specific antibody (Ab). The GBP-H1a fusion protein as a bioreceptor and the fluorescence-labeled Ab as a marker were used to provide an excellent detection signal. In addition, the chip fabrication and sensing characteristics are reported in detail.