An electrochemical impedance biosensor array with protein-modified electrodes was designed and fabricated in this report. To demonstrate its feasibility of the detection of multiple antigen-antibody binding reactions based on a label-free approach, human IgG (hIgG), rat IgG (rIgG), human globin and bovine serum albumin were immobilized, respectively, on the gold electrodes and then the resultant array was incubated with goat anti-hIgG, goat anti-rIgG, anti-human globin antibody and the mixture of three antibodies, respectively. The results indicated that the electron transfer resistance of the electrodes was significantly changed due to formation of the antigen-antibody conjugated layer. In addition, experimental conditions such as the protein concentration for the immobilization and screen were studied and optimized. Furthermore, the surface of various protein-modified electrodes was imaged with atomic force microscopy and the height distribution of protein particles was obtained with the Particle Analysis Software. The relative results were fully in accordance with the ones from the electrochemical impedance spectroscopy.