A simple and inexpensive method for fabricating a sensitive and compact indirect sandwich type electrochemical ELISA for the detection of estradiol using a chitosan electrodeposited platinum (Pt) wire microelectrode was proposed. In this assay, anti-17 beta estradiol antibody produced in goat (goat anti-estradiol Ab) was used as the capture antibody which was immobilized on the chitosan coated Pt wire microelectrode, anti-17 beta estradiol antibody produced in mouse (mouse anti-estradiol Ab) was used as the detection antibody, and goat anti-mouse IgG (immunoglobulin G) conjugated with alkaline phosphatase (AP) was used as the secondary antibody. The effect of pre-coated layers (chitosan, the capture antibody, and the blocking reagent BSA) on the electron transfer resistance (R-et) at the surface of ELISA electrodes has been investigated and analyzed by the electrochemical impedance spectrum (EIS). 4-Aminophenyl phosphate (4-APP) was chosen as the AP substrate and the oxidation potential of the electroactive AP product, 4-aminophenol (4-AP), on the Pt electrode was determined to be + 0.14 V (vs. Ag/AgCl). The electrochemical ELISA was detected by constant potential amperometry at + 0.14 V in the Tris buffer (pH 9.0). The limit of detection of this assay was 2.7 x 10(-1) pg/mL with a wide detection range from 2.7 x 10(-1) pg/mL up to 1.0 x 10(5) pg/mL. The assay specificity evaluated by testing the cross-reactivity of the assay for progesterone and 17 alpha-ethynylestradiol was found to be 0.033% and 3.4%, respectively. This assay has been tested with estradiol in spiked serum samples; however, further pretreatment of serum samples may be required to enhance precision and recovery. (C) 2015 Elsevier B.V. All rights reserved.