Heteroatom doping, particularly with nonmetallic atoms such as N, P, and S, has proven to be an effective strategy for modulating the fluorescent properties of carbon dots (CDs). However, there are few reports on the regulation of the photoluminescence of CDs by transition-metal doping. In this work, nickel-doped CDs (Ni-CDs) were fabricated using the hydrothermal approach. Ni atoms were incorporated into the sp² domains of the CDs through Ni-N bonds, resulting in an increased degree of graphitization of the Ni-CDs. Additionally, Ni-atom doping served to shorten the electron transition and recombination lifetimes, and suppress the nonradiative recombination process, resulting in an absolute fluorescence quantum yield of 54.7% for the Ni-CDs. Meanwhile, the as-prepared Ni-CDs exhibited excellent biocompatibility and were utilized for fluorescent bioimaging of HeLa cells. Subsequently, the Ni-CDs were employed as fluorescent anticounterfeiting inks for the successful encryption of two-dimensional barcodes. Our work demonstrates a novel heteroatom doping strategy for the synthesis of highly fluorescence-emitting CDs.