Given the significant impact of Fe3+ and ascorbic acid (AA) on human health, the development of an efficient sensor for their detection is of critical importance. In this study, a novel fluorescent probe composed of ultrathin boron nanosheets (Ultrathin BNSs) is introduced for the sensitive detection of both Fe3+ and AA in human serum. Ultrathin BNSs, exhibiting a fluorescence quantum yield of up to 6.8 % and minimal cytotoxicity, can be directly synthesized from boron through an ultrasound-assisted liquid-phase exfoliation (LPE) method. These Ultrathin BNSs demonstrate a selective and responsive fluorescence "on-off-on" mechanism toward Fe3+ and AA, governed by the induction and removal of an electron-transfer effect. The presence of Fe3+ induces non-radiative electron transfer, resulting in fluorescence quenching of the Ultrathin BNSs. In contrast, the introduction of AA facilitates the dissociation of Fe3+ from the Ultrathin BNSs/Fe3+ complex, leading to fluorescence recovery. When utilized as a fluorescent nanoprobe, Ultrathin BNSs exhibit a strong linear correlation with Fe3+ concentrations ranging from 0.2 to 150 µM and AA concentrations between 1.5 and 110 µM. Additionally, the limits of detection (LOD) for Fe3+ and AA reach 0.2 µM and 1.5 µM, respectively, demonstrating superior performance compared to numerous previously reported fluorescent probes. The feasibility of this Ultrathin BNSs-based sensor for AA detection in human serum was further examined, yielding satisfactory outcomes. It is expected that our strategy may offer a new approach for developing rapid, low cost and sensitive ultrathin BNSs-based sensors for biological sensing and environmetal applications.