A fluoro-microbead counting platform was developed to perform an optical immunoassay of various markers in real human samples. The fluoro-microbead guiding chip (FMGC) has four immunoreaction regions on a silicon oxide substrate, with five gold patterns imprinted on each region for multiple simultaneous assays. The FMGC assay clearly distinguished immunospecific binding from nonspecific binding by comparing optical signals from inside and outside of the patterns. To detect specific biomarkers, a sandwich immunoassay was performed using antibody-tagged fluoro-microbeads. The antigen-specific capture antibody was immobilized to the FMGC surface by reaction with 3-3'-dithiobis-propionic acid N-hydroxysuccinimide ester to create a self-assembling antigen-sensing monolayer (DTSP SAM) on the chip. A sample was applied to the antigen-sensing monolayer and allowed to react. To generate a binding signal, detection antibody-linked fluoro-microbead preparation step was added. The concentration of antigen such as cardiac troponin I (cTnI) and cartilage oligomeric matrix protein (COMP) in real human samples was determined by counting the number of biospecifically bound fluoro-microbeads on the corresponding five patterns on the FMGC. The optical signal showed a linear dependence with antigen concentrations in human samples such as blood and synovial fluid (SF). Optical detection and quantification of binding by fluorescence microscopy gave results that correlated well with results from a commercial ELISA for biomarkers in human plasma. Based on these findings, we propose that the FMGC-based immunoassay system may be adapted to detect and quantify a variety of clinically important targets in human samples.