Abstract: MoS₂-graphene nanocomposite was prepared. A novel electrochemical biosensor was constructed by immobilizing of aptamer on the electrode that was modified with Au nanoparticles and MoS₂-graphene. The electrochemical behavior of the modified electrode was tested by cyclic voltammograms, differential pulse voltammetric and electrochemical impedance. The reaction time and temperature between 17β-estradiol and aptamer were optimized. The experimental results showed that the proposed assay exhibited a good linear relationship between the peak current and the concentration of 17β-estradiol in the range of 1.0×10^-11~1.0×10^-8mol/L with the detection limit of 5.0×10^-12mol/L. The biosensor had good selectivity and stability