A novel biomaterial, polyglycerol grafted conducting polymer poly (3,4-ethylenedixythiophene) (PEDOT) was electrochemically synthesized. The PEDOT functionalized with hyperbranched polyglycerol (PEDOT-HPG) was simply prepared through the electrochemical polymerization of corresponding monomers, which was synthesized by grafting glycidol into (2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methanol (EDOT-MeOH). The antifouling properties of PEDOT-HPG surfaces were evaluated in terms of protein adsorption and the attachment of mammalian cells, including human cervical carcinoma cells (HeLa) and Michigan Cancer Foundation-7 (MCF-7). Through the conjugation of alpha-fetoprotein (AFP) antibodies to the PEDOT-HPG surfaces, sensitive immunosensors for AFP were fabricated, with the limit of detection at the level of 0.035 pg/mL. The prepared AFP biosensors exhibited excellent antifouling performance even in complex media and showed promising feasibility for the quantitative analysis of AFP in real samples. The PEDOT derivative with hyperbranched polyglycerol groups was effective to reduce biofouling and facile for surface functionalization. It is expected that this novel antifouling and conducting material may find broad applications in the development of electrochemical sensors and biosensors capable of assaying targets in complex biological media.