A screen-printed disposable electrode made of carbon ink modified with CuPc, P6LC and Nafion film is proposed as a low-cost and simple analytical method for sensitive determination of 17β-estradiol in different matrices. The electrochemical behaviour of 17β-estradiol was evaluated by cyclic voltammetry in the potential range from 0 to 0.55 V at a scan rate of 0.05 V s −1 using 0.1 mol L −1 phosphate buffer (pH 5.0) as supporting electrolyte. One oxidation peak was observed during anodic potential scanning, at a potential of 0.39 V. Under optimized experimental conditions, using the differential pulse voltammetry (DPV) technique, the proposed sensor showed a linear concentration range for 17β-estradiol between 8.0 × 10 −8 to 7.3 × 10 −6 mol L −1 , and detection limit of 5.0 × 10 −9 mol L −1 . The CuPc-P6LC-Nafion/SPE F sensor showed good selectivity, sensitivity, stability, reproducibility, and repeatability; it is a low-cost disposable device and shows no interference in DPV experiments. The proposed sensor was successful when applied to environmental and synthetic urine samples, with recoveries close to 100%.