The improvement of sensitivity of electrochemical immunosensor can be achieved via two approaches: increasing loading capacities of antibody and enlarging responding electrochemical signals. Based on these, CoFe2O4/graphene nanohybrid (CoFe2O4/rGO) as support was firstly used for preparing electrochemical biosensor, and with the addition of Au@Pd nanorods (NRs) as mimic enzyme, a label-free electrochemical immunosensor was prepared. Due to the high electrical conductivity, open porous structure and large loading capacities of CoFe2O4/rGO, the enhanced signal amplification between Au@Pd NRs and CoFe2O4/rGO was studied. Fabricated as a novel substrate, the prepared immunosensor had a good analytical performance and exhibited a wide linear range from 0.01 to 18.0 ng · mL(-1) with a low detection limit of 3.3 pg · mL(-1) for estradiol, which was succeeded in applying to detect estradiol in the natural water.