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Chronic exposure of 17β-estradiol (E2) even at low concentration can disorganize the endocrine system and lead to undesirable health problems in the long run. An electrochemical biosensor for rapid detection of E2 in water samples was successfully developed. The biosensor was based on split DNA aptamers attached onto poly (methacrylic acid-co-n butyl acrylate-succinimide) microspheres deposited on polypyrrole nanowires coated electrode (PPY/PMAA-NBA). The sandwich paired of split DNA aptamers used were truncated from 75 mer parent aptamers. These two strands of 12-mer and 14-mer split DNA aptamers were then immobilized on the PMAA-NBA microspheres. In the presence of E2, the split DNA aptamers formed an apt12-E2-apt14 complex, where the binding reaction on the electrode surface led to the detection of E2 by differential pulse voltammetry using ferrocyanide as a redox indicator. Under optimum conditions, the aptasensor detected E2 concentrations in the range of 1 × 10−4 M to 1 × 10−12 M (R2 = 0.9772) with a detection limit of 4.8 × 10−13 M. E2, which were successfully measured in a real sample with 97–104% recovery and showed a good correlation (R2 = 0.9999) with the established method, such as high-performance liquid chromatography. Interactions between short and sandwich-type aptamers (split aptamers) demonstrated improvement in aptasensor performance, especially the selectivity towards several potential interferents.