An aptamer-based electrochemiluminescent (ECL-AB) biosensor for ATP detection with high sensitivity and specificity was developed. The biosensor was assembled based on several steps. First, a complementary DNA (cDNA) of the ATP-binding aptamer, which has six complementary bases at both its ends, was hybridized with the aptamer molecule to form a rigid, linear double-stranded DNA (ds-DNA). The ds-DNA was then labeled with a ruthenium complex at the 3' terminus of cDNA, followed by the immobilization of this ds-DNA onto Au electrode surface through the 5'-HS on the cDNA. In the presence of ATP, due to the ATP binding to the aptamer, the aptamer molecules dissociated from the ds-DNA complex, which resulted in the formation of stem-loop structure of the single-stranded cDNA and led to the increase of the ECL signal. The increased ECL intensity was found linearly to the logarithm of the concentration of ATP ranging from 0.05 nM to 10 nM with a detection limit of 0.02 nM. Different from other ECL-AB biosensors with aptamers as the probes, this sensing system proposed here is based on the utilization of the cDNA of aptamers as the probes for ECL sensing. Therefore, such sensing system could provide a promising label-free and more readily regenerated model for aptamer-based small-molecules detection.