A polyurethane composite electrode modified with magnetic nanoparticles (mag; Fe3O4) coated with molecularly imprinted polymers (MIPs) was developed for the electrochemical determination of estradiol valerate. Chemical and morphological analyses of the mag-MIP were performed using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS), with the aim of characterizing this new material. The efficiency of adsorption of estradiol valerate by the mag-MIP was tested in binding experiments using an electrochemical method. For this, different electrodes (mag-MIP / graphite-epoxy composite (GEC), mag-non-imprinted polymer (NIP) / GEC, mag / GEC, and GEC) were evaluated by square-wave voltammetry (SWV). After 10 min at open circuit potential, increases in the current signal of 1.9, 2.1, and 3.0 times were obtained, comparing the electrochemical response of the mag-MIP / GEC sensor to the mag-NIP / GEC, mag / GEC, and GEC sensors, respectively. Under optimized conditions, the mag-MIP / GEC showed a linear concentration range for estradiol valerate of 5.0 × 10−7 to 7.5 × 10−4 mol L−1 and a limit of detection of 1.0 × 10−8 mol L−1. The proposed sensor was applied in the analysis of pharmaceutical, human urine, and river water samples. The recovery values determined using high performance liquid chromatography (HPLC)-UV and the electrochemical method were in agreement and were near 100%, demonstrating the reliability of the proposed method.