A label-free impedometric biosensor has been developed for the detection of estrogen hormone 17 beta-estradiol. The electrochemical biosensor has been fabricated by immobilizing both estrogen receptor-alpha and bovine serum albumin on gold electrode surfaces. The binding of 17 beta-estradiol to the estrogen receptors on the fabricated biosensor has increased the electron-transfer resistance which has been directly monitored by electrochemical impedance spectroscopy in the presence of 5.0 mM K3Fe(CN)(6)/K4Fe(CN)(6) (1:1, v/v) redox couple. The association constant between the 17 beta-estradiol and the estrogen receptors has been found to be ca. 1.8 x 10(11) M-1, which indicates that the estrogen receptors are well formed on gold electrode surfaces not only with high density but also with good specificity to its corresponding 17 beta-estradiol. The present biosensor gives a linear response (r(2) = 0.992) for 17 beta-estradiol concentration from 1.0 x 10(-13) to 1.0 x 10(-9) M with a detection limit of 1.0 x 10(-13) M (S/N = 3), which is much lower compared to those obtained with other detection methods. The present biosensor exhibits good stability, in which the biosensor has retained 88% of its initial activity after 3 weeks of storage in 50 mM phosphate buffer at pH 7.0. (C) 2012 Elsevier B.V. All rights reserved.