Using a biomedical sensor setup RIfS we have investigated the kinetic behavior of human albumin (Alb), human fibrinogen (Fib), and human immunoglobulin G (IgG) adsorbed onto surfaces of chitosan. Polystyrene (PS) was used as the control material in this study. The optical thickness of three kinds of proteins measured by RIfS was related to their molecular dimensions and potential orientations on a film surface. According to the operation principle of RIfS and the molecular dimensions of three kinds of proteins, the adsorbed layers of proteins onto the surface of chitosan and PS was calculated by using a newly introduced equation. The microstructure of the chitosan and polystyrene film and the surfaces with adsorbed proteins were imaged by atomic force microscopy (AFM). With AFM analyses the lateral distribution of the protein molecules on surfaces have been recognized. The results show that the number of adsorbed layers of the three proteins on the surface of chitosan are 0.635 for Alb, 0.158 for Fib and 0.0967 for IgG, and of polystyrene are: 0.577 for IgG, 0.399 for Fib, 0.336 for Alb. This study confirmed that RIfS is a useful tool for the analysis of plasma proteins adsorbed on a surface of biomaterials. Results show that at first on the surface of chitosan film much more Alb than Fib was adsorbed which demonstrated that chitosan has an antithrombus function. Secondly, on the surface of chitosan film more Alb and less Fib were adsorbed than on the surface of PS film, which demonstrated that chitosan has a better blood compatibility than polystyrene. Thirdly, the calculated layer number of the three proteins indicated that on both chitosan and PS substrates monolayer coatings form.