The aim of this study is (i) to prepare estrone-imprinted nanospheres (nano-EST-MIPs) and (ii) to integrate them into the electrochemical sensor as a recognition layer. N-methacryloyl-(l)-phenylalanine (MAPA) was chosen as the complexing monomer. Firstly, estrone (EST) was complexed with MAPA and the EST-imprinted poly(2-hyroxyethylmethacrylate-co-N-methacryloyl-(l)-phenylalanine) [EST-imprinted poly(HEMA-MAPA)] nanospheres were synthesized by surfactant-free emulsion polymerization method. The specific surface area of the EST-imprinted poly(HEMA-MAPA) nanospheres was found to be 1275 m(2)/g with a size of 163.2 nm in diameter. According to the elemental analysis results, the nanospheres contained 95.3 mmole MAPA/g nanosphere. The application of EST specific MIP nanospheres for the development of an electrochemical biosensor was introduced for the first time in our study by using electrochemical impedance spectroscopy (EIS) technique. This nano-MIP based sensor presented a great specificity and selectivity for EST.