Abstract: Estradiol (E2) was bound to bovine serum albumin (BSA) to form a complete antigen (E2-BSA), after the hydroxy (-OH) at the position of C3 was activated. A BALB/c mouse was immunized with the mixture of E2-BSA and Freund adjuvant. Monoclonal antibody-producing hybridoma anti-E2 was prepared by fusion of SP2/0 murine myeloma cells with spleen cell isolated from the immunized mouse. A monoclonal antibody (McAb) specific for E2 was produced in vivo by the hybridoma anti-E2. Analysis revealed that anti-E2 McAb (E2-McAb) was of the IgG2a type, with the molecular weight179000 daltons. The titer of E2-McAb was 1:106, and the affinity constant of constant of E2-McAb was 2.9×108L/mol.