A convenient electrochemical sensing pathway for 17β-estradiol detection was investigated. The system is based on a conducting polymer and horseradish peroxidase (HRP) modified platinum (Pt) electrode. The miniature estradiol biosensor was developed and constructed through the immobilization of HRP in an electroactive surface of the electrode covered with electroconducting polymer – poly(4,7-bis(5-(3,4-ethylenedioxythiophene)thiophen-2-yl)benzothiadiazole). The detection strategy is based on the fact that 17β-estradiol (E2) and pyrocatechol (H₂Q) are co-substrates for the HRP enzyme. HRP, which does not react with E2, in the presence of H₂O₂ catalyses the oxidation of H₂Q to o-benzoquinone (Q). With the optimized conditions, such constructed biosensing system demonstrated a convenient level of sensitivity, selectivity in a broad linear range – 0.1 to 200 μM with a detection limit of 105 nM. Furthermore, the method was successfully applied for hormone detection in the presence of potential interfering compounds (ascorbic acid, estriol, estrone, uric acid and cholesterol).