The invention discloses a preparation method of an estradiol magnetic molecularly imprinted nanoparticle electrochemical sensing membrane. According to the preparation method, an estradiol magnetic molecularly imprinted polymer is obtained via preparation of a molecularly imprinted self-assembled solution, polymerization, and template molecule elution. The estradiol magnetic molecularly imprinted nanoparticle electrochemical sensing membrane is high in selectivity, response speed, sensitivity, stability, tolerability, and universality, and is capable of realizing high efficiency, sensitive, real-time detection of estradiol in composite matrix.