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The remarkable features of a carbon fiber paper sensor (CP) were employed for detection of the estrogenic hormone 17α-ethinylestradiol (EE2), considered a contaminant of emerging concern due to its potential ecotoxicity and widespread in the aquatic ecosystems. In this work, an unpreceded CP pre-treatment study was conducted with the (Il)-hexacyanoferrate(III) ion pair, however a bare sensor without pre-treatment revealed higher efficiency on the oxidation of EE2 compared to a chemical and electrochemical pre-treated CP and a gold nanoparticles modified CP, being thus selected for EE2 determinations. The analytical conditions were thoroughly optimized in terms of electrolyte pH (pH 7), differential pulse voltammetry parameters (modulation time 0.003 s, amplitude 0.09 V, interval time 0.1 s and step potential 0.01 V), and analyte preconcentration potential (0.4 V) and time (180 s). The hormone can be determined by the CP in a wide linear range from 0.1 to 1000 nM, achieving a detection limit of 0.14 ± 0.005 nM and an outstanding sensitivity of 1636 ± 232 μA μM−1 cm−2 in the lowest linear zone (0.1–1 nM). The sensor was validated in river water and fish reaching good recoveries (91.2 ± 4.6 to 109.0 ± 7.1%), reproducibility and repeatability. Moreover, the sensor showed high selectivity to EE2 in the presence of several potential interfering compounds and frequently prescribed drugs, though it could not discriminate the similar hormone, 17β-estradiol, being the total concentration obtained in this case. CP-based sensors emerge as efficient electroanalytical tools, suggesting that modification of the surface may not always be beneficial in terms of sensitivity. [Display omitted] • A simple and effective carbon paper sensor was used for 17α-ethinylestradiol sensing. • The best analytical signal was obtained with an untreated and unmodified carbon paper. • The optimized sensor achieved high sensitivity (1636 μA μM−1 cm−2) and LOD of 0.14 nM. • Application in complex samples of fish resulted in recoveries from 102 to 109%. • Carbon paper sensors can be become valuable tools in the electroanalytical field. [ABSTRACT FROM AUTHOR]