Developing reliable and sensitive detection methods for adenosine triphosphate (ATP) is vital for both clinical diagnosis and food safety. In this work, by coupling aptazyme- and catalytic hairpin assembly (CHA)-based signal amplification and electrochemiluminescence (ECL), an ultrasensitive biosensor for sensing ATP was fabricated using Ru(bpy)32+-doped silica nanoparticles (RuSiO2) as ECL probes and a ferrocene-functionalized hairpin DNA (hairpin-Fc) as quencher. The aptazyme-triggered cleavage of the DNA substrate and the CHA reaction both led to the circular release of trigger DNA, resulting in a significant dual signal amplification, with unprecedented enhancement up to 940-fold. Moreover, the bioconjugation of the DNA substrate with Au@Fe3O4 facilitated the separation and purification of the released trigger DNA, and effectively reduced the background signal. As a result, the as-prepared ECL biosensor exhibited a much lower detection limit of 0.054 pM for ATP, compared to those in previous reports, and showed high reliability for ATP detection in both spiked serum samples and Staphylococcus aureus. This work offers a new perspective for designing nucleic acid-based signal amplification for detecting ATP in bacterial analysis and clinical diagnosis.