A simple and label-free electrochemical aptasensor was developed for detecting 17β-estradiol (E2). To translate the binding events between aptamer and E2 into the measurable electrochemical signal, the nickel hexacyanoferrate nanoparticles (NiHCF NPs) as signal probe was in situ introduced on the electrode by a simple two-step deposition method, exhibiting well-defined peaks with good stability and reproducibility. Subsequently, Au nanoparticles (Au NPs) was covered on the NiHCF NPs, which not only provided a platform for immobilizing the aptamer by S-Au interaction, but further enhanced the conductivity and stability of the signal probe. With the addition of E2, the formation of E2-aptamer complexes on the sensing interface retarded the interfacial electron transfer reaction of the probe, resulting in the decrease of the electrochemical signal. E2 could be readily examined by measuring the signal change. A linear range of 1×10(-12)-6×10(-10) M was obtained with a low detection limit of 0.8×10(-12) M. The aptasensor also exhibited high specificity to E2 in control experiments employing seven endocrine disrupting compounds as the interferents that had similar structure or coexisted with E2 in the environment. Besides, the applicability of the aptasensor was successfully evaluated by determining E2 in the real samples.