In this work, an electrochemiluminescent (ECL) biosensor was constructed on the basis of amino-modified 3,4,9,10-perylenetetracarboxylic dianhydride/luminol (PTC-NH2/Lu) nanocomposite as emitter and bipedal DNA walker signal amplification strategy for ultrasensitive detection of microRNA-21 (miRNA-21). The PTC-NH2/Lu nanocomposite was prepared as signal tag via π-π stacking molecular assembly, in which amino-modified 3,4,9,10-perylenetetracarboxylic dianhydride (PTC-NH2) as a novel coreaction accelerator significantly enhanced the ECL emission of luminol-H2O2 system. Moreover, target miRNA-21 triggered bipedal DNA walker was powered by toehold-mediated strand displacement reaction (TSDR) for signal amplification. Consequently, the proposed ECL biosensor achieved ultrasensitive detection of miRNA-21 with a linear range from 100 aM to 100 pM and a limit of detection of 33 aM. Simultaneously, the biosensor was also successfully applied to detect target miRNA-21 in lysates from human cancer cells. As a result, this work constructed a new signal amplification platform, exhibiting great application potential in biomedical analysis and early clinical diagnostics.