A novel strategy for the fabrication of electrochemical aptasensor is proposed in this work, and the strategy has been employed to develop an aptasensor for the assay of adenosine deaminase activity. While a well-designed oligonucleotide containing three functional regions (an adenosine aptamer region, a G-quadruplex halves region, and a linker region) is adopted in our strategy as the core element, the enzymatic reaction of adenosine catalyzed by adenosine deaminase plays a key role as well in the regulation of the binding of the G-quadruplex halves with hemin, the electroactive probe, which is to reflect the activity of the enzyme indirectly but accurately. The detection limit of the fabrication biosensor can be lowered to 0.2 U mL(-1) of adenosine deaminase, and 1 nM of the inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride is enough to present distinguishable electrochemical response. Moreover, since the electroactive probe is not required to be bound with the oligonucleotide, this strategy may integrate the advantages of both the labeled and label-free strategies.