Estradiol levels in women are related to lung, uterine (endometrial), ovarian and breast cancers, although the exact mechanism of cancer development is not fully understood. A new cost-effective sensor was fabricated by incorporating conductive NiFe2O4 metal oxide into the electrochemical sensitive platform of mesoporous carbon (MC) derived from cotton to measure estradiol determination by electrochemical methods. A new modified GCE/NiFe2O4-MC electrode, which exhibits electrocatalytic activities, was created by dropping NiFe2O4-MC onto the glassy carbon electrode (GCE) surface using the drop casting technique to detect the electroactive estradiol molecule. Estradiol was determined electrochemically by using the square wave voltammetry technique with a detection limit of 6.88 nM in 20.0-566 nM linear range in phosphate buffer solution at pH 7.4 with this novel modified GCE/NiFe2O4-MC electrode, that has the best electron transfer rate. The simultaneous determination of estradiol in the presence of testosterone was successfully performed in 0.1 M tetrabutylammonium tetrafluoroborate supportive electrolyte solution prepared in acetonitrile, an anhydrous medium. Real sample applications were carried out to prove the applicability and precision of the novelty-produced electrode. The amount of estradiol in tablet samples was obtained satisfactorily with high recovery values by standard addition method. (C) 2020 The Electrochemical Society ("ECS"). Published on behalf of ECS by IOP Publishing Limited.