In this work, we presented a novel label-free biosensor for rapid detection of bleomycinsulphate (BLM). The biosensor was based on the fluorescent “turn off-on” of nitrogen-doped graphene quantum dots (N-GQDs), which was prepared in a green way from citric acid and ammonia. The richness of carboxyl groups on the N-GQDs enabled strong adsorption of ssDNA to the surface of N-GQDs through π-π stacking interactions, resulting in the effective fluorescence quenching of N-GQDs system. The ssDNA underwent an irreversible cleavage event via the oxidative effect of BLM with Fe(II) as a cofactor, thus a turn-on fluorescence signal was observed. Thereby, the concentration of BLM can be quantitatively determined in a broad range from 0.34 nmol/L to 1300 nmol/L with a detection limit of 0.34 nmol/L. The presented method was applied to the determination of BLM in human serum samples with satisfactory results. •A novel label-free fluorescent sensor for BLM has been established.•The oxidative effect of BLM-Fe(II) on ssDNA was adopted in the detecting strategy.•The proposed method exhibited a wide linear range, low detection limit, good selectivity, and anti-interference ability.•The assay of BLM in human serum samples was realized with satisfactory results.