Engineered nanoparticles adsorb proteins in biological fluids, forming a dynamic protein corona. This protein corona may alter the biological identity of the nanoparticles, thus affecting their cellular pathways. However, the thorough understanding of protein corona formation is lacking due to the insufficiency of current assemble techniques and low-temporal resolution microscopic imaging methods. Here, we have developed an optical method with real-time single particle tracking system, to first study individual free diffusing polystyrene nanoparticles interacting with bovine serum albumin proteins. The size measurement and tightly-adsorbed protein quantification of each nanoparticle-protein complex showed strong heterogeneity in protein adsorption. Our findings will provide an insight for the physiological application and cytotoxic study of the engineered nanoparticles in vivo.