In this study, we report a new immunoassay platform using yeast cell surface display. This method holds promise for very low limit of detection (LOD) and is suitable for 2-Plex antibody recognition. Instead of adopting a conventional enzyme linked immunosorbent assay (ELISA) protocol by detecting the enzymatic activities or other physicochemical properties of the labeled analytes, this approach determines the quantity of an antibody analyte by directly counting the amount of "modified" yeast cells bound with antibody on the cell surface. c-myc and hemagglutinin (HA) tags were employed as an epitope model to demonstrate our approach. This yeast surface display based cell counting immunoassay (abbreviated as YSD-CCI) for anti-c-myc has a detection limit of 0.2 ng/mL, which is about 80 times higher than that of a conventional yeast ELISA under a similar condition. Moreover, the YSD-CCI's capability for 2-Plex antibody detection was demonstrated by simultaneous detection of anti-c-myc and anti-HA using engineered yeast cells expressing intracellular enhanced green fluorescent protein (EGFP) and mCherry, respectively. This proof-of-concept study paves the way for a new ultrasensitive multiplexed immunoassay method for diagnostic applications.