The invention provides a method for preparing a BCNO light-emitting nanosheet. The method includes the following steps that firstly, boric acid and dicyandiamide are placed in an oven for heating pretreatment, heating temperature is 110-130 DEG C, heating time is 4-6 hours, and boric acid and dicyandiamide are naturally cooled to room temperature after being heated; secondly, boric acid and dicyandiamide subjected to heating pretreatment are immediately placed in a reactor, then ethylene glycol is added, solution temperature is raised after stirring to dry the solution by distillation, and a precursor of the BCNO nanosheet is obtained; thirdly, the precursor, prepared in the second step, of the BCNO nanosheet is sintered at the temperature of 600-700 DEG C, the temperature increase rate is 5 DEG C/min, sintering time is 4-6 hours, the precursor is naturally cooled to room temperature, and the BCNO light-emitting nanosheet is obtained. The BCNO light-emitting nanosheet synthesized through the method is free of toxin and pollution, the emitting range is a blue and green light wave band, and the BCNO light-emitting nanosheet can serve as fluorescein to be used in the fields such as cell labeling, cell imaging and biomedicine.