A series of rigid macroporous polymer layers differed by hydrophobic–hydrophilic properties was synthesized in situ in preliminary fabricated wells and applied as the platforms for protein biochips. Scanning electron microscopy, etalon porosimetry and BET analysis were used for materials characterization. The comparison of analytical efficiency of the developed platforms allowed for the choice of the most optimal polymer, as well as the evaluation of impact of material porous properties. The quantitative parameters of affinity interaction between two different protein pairs were calculated depending on biochip characteristics using the developed analytical protocol. Moreover, the described biochips were successfully tested to detect acetylcholinesterase via catalytic reaction followed by the formation of fluoresceine as a product. Different parameters of enzymatic reaction were calculated for the reaction on a chip and compared to those established for in solution process.