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A surface plasmon resonance (SPR) based immunosensor is presented for highly sensitive and selective detection of 17β-estradiol by the indirect competitive inhibition immuno assay, employing anti-17 β-estradiol antibody as high molecular weight (HMW) interactant. Immobilization of estradiol-BSA conjugate onto the nano thin gold surface was accomplished by covalent amide linkage through self assembled monolayer. The proposed biosensor is simple to fabricate, reproducible and exhibit excellent sensitivity for estrogen (detection limit,1 pg mL−1) without any significant interference from structurally similar steroidal hormone, progesterone and non-steroidal compound bisphenol-A. The proposed surface displayed a high level of stability during repeated regeneration and immunoreaction cycles suitable for biosensor development.