It was found that tripropylamine (TPA) could be used as a coreactant to initiate the electrochemiluminescence (ECL) of acridinium NHS ester (AE NHS) labels attached to DNA. The radicals generated in the electro-oxidation process of TPA reacted with AE NHS to form the excited N-methylacridone, giving rise to light emission. The AE/TPA ECL system was for the first time used as the detection system for developing an ECL-based DNA sensor. In the protocol, streptavidin-modified gold nanoparticles were firstly immobilized onto a thiol-treated gold electrode. The streptavidin could specifically interact with the biontinylated capture DNA. Afterwards, the target DNA and the AE-labeled report DNA were conjugated onto the electrode step by step due to the hybridization reactions, and a sandwich-type sensor was fabricated. The ECL signals of the sensor were obtained under pulse potential condition in alkaline solution containing 50.0 mmol L(-1) TPA. Under optimized experimental conditions, the linear range of the DNA sensor for the determination of the target DNA was from 5.0 × 10(-15) to 5.0 × 10(-12) mol L(-1). The detection limit (S/N = 3) was 3.0 × 10(-15) mol L(-1). Moreover, the sensor could specifically recognize the target DNA against one base-pair mismatched sequences, two base-pair mismatched sequences, and the noncomplementary sequences. It is of great application potential in clinic analysis.