In this paper, we present a new microfluidic immunoassay platform, which is based on the synergistic combination of the yeast surface display (YSD) technique and the microfluidic technology. Utilizing the YSD technique, antigens specific to the target antibody are displayed on the surface of engineered yeast cells with intracellular fluorescent proteins. The displayed antigens are then used for the detection of the target antibody, with the yeast cells as fluorescent labels. Multiplex immunoassay can be readily realized by using yeast cells expressing different intracellular fluorescent proteins to display different antigens. The implementation of this YSD-based immunoassay on the microfluidic platform eliminates the need for the bulky, complex and expensive flow cytometer. To improve the detection sensitivity and to eliminate the need for pumping, a functionalized micro pillar array (MPA) is incorporated in the microfluidic chip, resulting in a detection limit of 5 ng/mL (or 1 ng in terms of amount) and enhanced compatibility with practical applications such as clinical biopsy. This new platform has a high potential to be integrated into microfluidic detection systems to enable portable diagnostics in the future.