Poly(3,4-ethylenedioxythiophene) (PEDOT) has been regarded as one of the most attractive conductive materials for making bioelectronics. Surface modification is one of the most important methods to enhance the performance of PEDOT-based bioelectronics. To investigate the surface charge effect on the behavior of protein binding on PEDOT, we develop a new positively charged functionalized EDOT monomer, EDOT-N+, to copolymerize with another negatively charged carboxylic acid-modified EDOT monomer, EDOT-COOH. The poly(EDOT-N+-co-EDOT-COOH) film is prepared by the electropolymerization method. By mixing the two monomers with different feed ratios, we can tune the distribution of surface charges. The actual composition of the copolymer is determined by toluidine blue O staining. Based on the result of TBO staining, a copolymer with a 1:1 ratio of EDOT-N+ to EDOT-COOH is obtained when the monomer solution of composition at [EDOT-N+]: [EDOT-COOH] = 8 : 2, which is due to a slower polymerization rate of EDOT-N+ compared to EDOT-COOH. To further testify the antifouling property of poly(EDOT-N+-co-EDOT-COOH), nonspecific protein binding of bovine serum albumin and lysozyme were measured in situ by using a quartz crystal microbalance. The results also show that at [EDOT-N+]: [EDOT-COOH] = 8 : 2, poly(EDOT-N+-co-EDOT-COOH) presented good antifouling property. Therefore, we propose a zwitterion-like antifouling property from the poly(EDOT-N+-co-EDOT-COOH) with an equal density of both positive and negative functional groups.