In this paper, we propose a fluorescent biosensor for the sequential detection of Pb 2+ ions and the cancer drug epirubicin (Epn) using the interactions between label-free guanine-rich ssDNA (LFGr-ssDNA), acridine orange (AO), and a metal-phenolic nanomaterial ( i.e. , nano-monoclinic copper-tannic acid (NMc-CuTA)). An exploration of the sensing mechanism shows that LFGr-ssDNA and AO strongly adsorb on NMc-CuTA through π-π stacking and electrostatic interactions, and this results in the fluorescence quenching of AO. In order to sense the target Pb 2+ , initially, LFGr-ssDNA specifically binds with Pb 2+ ions to form a G4 complex (G-Pb 2+ -G base pair), which was released from the surface of NMc-CuTA with strong AO fluorescence enhancement (Turn-ON). The subsequent addition of a biothiol, like cysteine (Cys), to the G4 complex decreases the fluorescence, as the Pb 2+ ions released from the G4 complex have a higher interaction affinity with the sulfur atoms of Cys; this further induces the unwinding of the G4 complex to form LFGr-ssDNA. Finally, Epn was added to this, which intercalates with LFGr-ssDNA to form a G4 complex via G-Epn-G, resulting in fluorescence recovery (Turn-ON). Accordingly, the Turn-ON fluorescent probe had subsequent limits of detection of 1.5 and 5.6 nM for Pb 2+ and Epn, respectively. Hence, the reported NMc-CuTA-based sensing platform has potential applications for the detection of Pb 2+ and Epn in real samples with good sensitivity and selectivity. In this paper, we propose a fluorescent biosensor for the sequential detection of Pb 2+ ions and the cancer drug epirubicin (Epn) using the interactions between label-free guanine-rich ssDNA (LFGr-ssDNA), acridine orange (AO), and a metal-phenolic nanomaterial.