Rapid assessment of acute myocardial infarction (AMI) was successfully demonstrated using an improved superparamagnetic polymer microsphere-assisted sandwich fluoroimmunoassay to detect two early cardiac markers-myoglobin and human heart-type fatty acid binding protein (H-FABP). This assay used a preparation of superparamagnetic poly(styrene-divinylbenzene-acrylamide) microspheres, glutaraldehyde-coupled capture antibodies (monoclonal anti-myoglobin 7C3 and anti-H-FABP 10E1) grafted onto the polymer microspheres, and a sequential sandwich fluoroimmunoassay using detection antibodies (FITC-labeled anti-myoglobin 4E2 and FITC-labeled anti-H-FABP 9F3). Characterization of the polymer microspheres by TEM, SEM and Fourier transform infrared spectroscopy (FT-IR) showed that the microspheres were uniformly round with an average diameter of 1.12 m, and had a Fe3O4-polymer core-shell structure (shell thickness was about 84 nm) with 0.22 mmol/g amino groups on their surfaces. The magnetic behavior of the Fe3O4-polymer microsphereswas superparamagnetic (Ms=13 emu/g, Hc=13.1 Oe). Fluorescence images of the post-immunoassay microspheres recorded using a confocal laser-scanning microscope showed that the average fluorescence intensity was correlated with the concentration of cardiac markers, in agreement with the results obtained by an F-4500 FL spectrophotometer; this indicated that the fluoroimmunoassay could be used to semi-quantitatively detect both myoglobin and H-FABP. The detection limit was 25 ng/mL for myoglobin and 1 ng/mL for H-FABP.