A reusable electrochemical aptasensor for highly sensitive detection of small molecules had been developed using adenosine as a model molecule. The sensing interface was fabricated by self-assembling the part DNA duplex hybridized by T-thiolated part complementary strand (TPCS) and 3'-ferrocene(Fc)-labeled adenosine-binding aptamer strand (FABA) through S-Au bonding on a gold electrode surface. When the modified electrode was incubated in the adenosine solutions, the aptamer made structure switching to bind adenosine. As a result, Fc-labeled adenosine-binding aptamer strand was taken off from the sensing interface, resulting in a decrease of the redox current. The aptasensor was characterized electrochemically by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The CV of the resulting aptasensor showed a linear response to the increase of the adenosine concentration in the range of 5.0 x 10(-8)-1.5 x 10(-5) M With a linear correlation of r = 0.9898 and a detection limit of 1.65 x 10(-8) M. Moreover, the aptasensor exhibited several excellent characteristics, such as high sensitivity, selectivity, good stability, and reproducibility. (C) 2009 Published by Elsevier Ltd.