A carboxylated superparamagnetic microbead-assisted sandwich fluoroimmunoassay was successfully demonstrated for the analysis of the early protein markers, myoglobin and human heart-type fatty acidbinding protein (H-FABP), associated with acute myocardial infarction. This assay approach consisted of the preparation of superparamagnetic polymer microbeads using a dispersion polymerization, followed by grafting of capture antibodies (monoclonal anti-H-FABP 10E1 and anti-myoglobin 7C3) onto the polymer microbeads using EDC-NHS protocol, and then a sequential sandwich fluoroimmunoassay using detection antibodies (FITC-labeled anti-H-FABP 9F3 and FITC-labeled anti-myoglobin 4E2). The Fe3O4 nanoparticles and carboxylated Fe3O4-polymer microbeads were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectrophotometry, vibrating sample magnetometry, and X-ray diffraction. The fluoroimmunoassay images were recorded using a confocal laser-scanning microscope, and the average fluorescence intensity of the microbeads was found to correspond to the concentration of each cardiac marker, in agreement with the results obtained by a spectrofluorophotometer. The carboxylated magnetic microbead-assisted protocol could be utilized to semi-quantitatively detect both myoglobin and H-FABP.