Graphdiyne (GDY) is an sp and sp2 co-hydrocarbon allotrope whose particular structure endows it with many fascinating properties, including abundant chemical bonds, high conjugation, natural pores, high carrier mobility, high conductivity and stability, etc. In this work, two-dimensional graphdiyne is prepared as an electrode substrate material coupling with an exonuclease III-assisted amplification strategy to construct a superior-performance self-powered biosensor based on enzymatic biofuel cells for highly sensitive detection of the tumour marker miRNA-21. Glucose oxidase (GOD) is first immobilized on the GDY/AuNP composite to prepare a bioconjugate. GDY/AuNP modified carbon cloth is used as an enzyme biofuel cell electrode, which is then modified with bilirubin oxidase as a biocathode. The bioconjugate binds to GOD through specific binding to the bioanode. When miRNA-21 is present, specific recognition by exonuclease III in the system results in cleavage of the capture probe, and miRNA-21 is recovered and involved in the cycle. The target miRNA-21 then causes corresponding changes in the open-circuit voltage of the self-powered system. Based on this, a sensitive detection method was constructed, within the scope from 0.1 fM to 0.1 nM with a shallow detection limit of 55.2 aM (S/N = 3). The new approach triumphantly has been used to detect miRNA-21 in serum, which provides a compelling new way for early diagnosis of related cancers.