The understanding of biomolecular interactions on solid surfaces is of importance for the design of new biomaterials and medical devices. In this work, the adsorption behavior of creatine phosphokinase (CPK) onto hydrophilic (silicon wafers and amino-terminated surfaces), hydrophobic (polystyrene), and charged (sulfonated polystyrene films) substrates was investigated by means of in situ ellipsometry, contact angle measurements, and atomic force microscopy. CPK is an interesting biomolecule due to its large application in the diagnosis for myocardial infarction and muscle disorders. In the dilute regime (c similar to 0.005 g/L) the ellipsometric measurements revealed that the kinetics adsorption process of CPK onto silicon wafers and amino-terminated surfaces can be divided into four stages: (i) a diffusive one, (ii) adsorption and rearrangement, (iii) formation of a monolayer, and (iv) continuous and irreversible adsorption caused by relaxation process and cooperative binding. This seems to be the first time that such a behavior has been experimentally observed. For more concentrated solutions, the CPK formed aggregates in solution and, therefore, the adsorption increased continuously with time. CPK adsorbed irreversibly either on hydrophilic or on hydrophobic substrates. The adsorption isotherms showed a preferential adhesion of CPK onto the hydrophilic substrates. Since hydrophilic segments predominate the CPK structure, hydrogen bonding seems to play a major role in the adsorption process.