Apart from the huge physiological importance of hemo-, myo- globin and hemin, they play also an important role as robust and effective recognition agents in chemical and biological sensors. In this aspect the key factor in developing a reliable biosensor is the immobilization on the transducer. Usually immobilization can be produced by chemical methods, but there is frequently a need for homogeneous films of well-controlled thickness or films which can be deposited in a dry environment. The film thickness is of main importance for the optical transducer detected by Surface Plasmon Resonance (SPR) what is used in our study. Hundreds of immobilization protocols have been developed in an effort to ensure high performance sensing. All of them are focused on finding and deposition of appropriate matrices in which the recognition medium can be incorporated. However, the matrix always deteriorates the effectiveness of recognition. It seems that the best approach is to perform direct immobilization of the recognition medium. However, this is not always possible regarding the organic materials – the problem is whether the deposition retains the bioactivity of the recognition agent. On the other hand, the type of the transducer also imposes constrains. For example, the direct immobilization of the proteins is not possible for electrochemical sensors, because of the distance between the redox center and electrodes is too long. Evaluating the pros and cons of organic (protein) film deposition we have considered to study the possibility for direct immobilization of myoglobin, hemoglobin and hemin on SPR transducer. To best of our knowledge, SPR biochip with immobilized myoglobin, hemoglobin and hemin has never been constructed before. We have used spin coating, for direct immobilization and matrix-assisted pulsed-laser evaporation (MAPLE) for elaboration of the SPR biochip. The performance of both SPR chips – direct and MAPLE immobilized, was studied by SPR registration of the binding activity of myo- and hemo- globin ligands with carbon monoxide (CO), carbon dioxide (CO<sub>2</sub>) and nitride oxide (NO).