Enzyme-linked immunosorbent assays (ELISA), one of the most common immunoassays, is widely used for detection and quantification of chemical and biological molecules and is becoming more and more important in clinical diagnostics. To reduce the experimental time and reagent quantities, a polymeric microfluidic biochip has been designed and fabricated based on the integration of manufacturing modules: polyaniline-based surface modification, the conceptual design with splitters, and the oxygen plasma-PEI-TR protein A modification. The proper flow sequencing was achieved on a CD-like microfluidic chip by integrating the necessary microfluidic functions such as capillary valving treated with polyaniline-based surface modification. It has been successfully demonstrated that the splitters indeed work very well to even distribution of the fluid to branch flows. To enhance the sensitivity of miniature detection area on the PMMA biochips for ELISA immunoassay, a TR-catalyzed protein A antibody immobilization technique was successfully developed. By using this method, not only the antibody binding efficiency, but also the specific capture capacity towards target protein of the antibody was greatly enhanced, leading to a significant improvement of the fluorescence signal ELISA in PMMA chips including a lower detection limit, a wider linear detection range, a much shorter assay time comparable to 96-well microtiter plates. The microfluidic biochips we developed here would be applicable to a variety of clinically relevant disease conditions. And the modification technologies in this study can be extensively implemented in lab-on-a-chip systems, drug/gene delivery carriers and other immunoassay biosensor applications.