Amonafide, an anthracene derivative, exerts anticancer effects through DNA intercalation and topoisomerase II inhibition, but its clinical use is limited by low efficacy and severe side effects. To address these limitations, we designed biomarker-responsive amonafide-based prodrugs for selective activation in tumor microenvironments enriched with nitroreductase (NTR) or hydrogen sulfide (H 2 S). Among them, Amo-c-NO 2 exhibited the most potent anticancer activity and selective fluorescence activation at 585 nm in response to NTR, enabling real-time tumor imaging, in both live cells and 3D tumor spheroid models. Once activated, Amo-c-NO 2 translocates from mitochondria to the nucleus, inducing apoptosis via topoisomerase inhibition and DNA damage. In vivo , it effectively suppressed tumor growth with minimal side effects. These findings establish Amo-c-NO 2 as a promising biomarker-activated theranostic agent for precise and effective cancer therapy. • Rational design and synthesis of a series of biomarker-responsive amonafide-based prodrugs. • Amo-c-NO 2 is selectively activated by nitroreductase (NTR) under tumor hypoxia. • NTR-activated Amo-c-NO 2 induces apoptosis via mitochondrial-to-nuclear translocation and DNA damage. • Amo-c-NO 2 demonstrates potent anticancer activity while sparing normal cells, in both in vitro and in vivo . • Amo-c-NO 2 effectively suppresses tumor growth with minimal side effects in xenograft models.