A new flow-through electrochemical aptasensor was designed for ultrasensitive monitoring of adenosine triphosphate (ATP) by coupling microvalve-programmable capillary column with CdS-functionalized DNA concatamer for signal amplification. Initially, a layer of primary DNA-conjugated polyacrylamide hydrogel was covalently linked onto the internal surface of capillary column, and then an automated sequenctial injection format with a syringe pump was employed for development of the programmable capillary-based aptasensor. In the presence of target DNA aptamer, the immobilized primary DNA hybridized with partial bases of the aptamer. The excess aptamer fregment could trigger the formation of DNA concatamer between auxiliary DNA1 and CdS-labeled auxiliary DNA2. Upon target ATP introduction, a specific ATP-aptamer reaction was excuated, thereby resulting in the release of CdS-functionalized DNA concatamer from the capillary. Subsenquent anodic stripping voltammetric detection of cadmium released under acidic conditions from the released CdS nanoparticles could be conducted in a homemade detection cell. Under optimal conditions, the dynamic concentration range spanned from 0.1 pM to 10nM ATP with a detection limit of 0.06 pM ATP. The electrochemical aptasensor showed good reproducibility, selectivity, and stability. In addition, the methodology was evaluated for the analysis of ATP spiked serum samples, and the recoveries was 81-140%.