Various polymeric surfaces (polyester, polyethylene, polystyrene) were functionalized under oxygen and dichlorosilane-RF-cold-plasma environments and were employed as substrates for further in situ derivatization reactions and immobilization of alpha-Chymotrypsin. The nature and morphology of the derivatized substrates and the substrates with immobilized enzymes were analyzed using survey and high-resolution X-ray photoelectron spectroscopy, attenuated total reflectance-fourier transform infrared (ATR-FTIR), laser desorption fourier transform ion cyclotron resonance mass spectrometry, chemical derivatization, and atomic force microscopy (AFM) techniques. It was demonstrated that the tacticity of the polystyrene substrate did not notably influence the activity of the immobilized enzyme, however, spacer molecules intercalated between the polymeric substrates (e.g., polyethylene) and the enzyme significantly increased the enzyme activity (comparable with that of the free enzyme). Computer-aided conformational modeling of the substrate-spacer systems indicated that the longer the spacer chain, the greater the mobility of the enzyme. It is suggested that the greater mobility of the enzyme molecules is responsible for the enhanced activity. It has also been shown that the stability of the immobilized enzyme systems was good; they retained their activity during several washing/assay cycles.