The study of protein adsorption on solid surfaces is interesting for theoretical and practical bio‐analytical sensing applications. In this work we combine electrochemical impedance spectroscopy, enzyme linked immunosorbent assay, and fluorescence microscopy with thin boron doped nanocrystalline diamond films to address and study the adsorption behavior of globular proteins (antibodies) on hydrophobic and hydrophilic diamond surfaces. A powerful combination of time resolved impedance spectroscopy and data modeling with equivalent circuits allow a detailed insight in the protein behavior at an interface. It is found that hydrogenated diamond is greatly favorable for impedimetric read‐out but causes slight conformational loss of the protein structure and therefore also its biological activity. The oxidized surface allows faster adsorption and a high biological activity but results in smaller impedimetric response.