The invention relates to preparation and application of a fluorescent carbon dot nanoprobe for detecting methylene blue and living cell imaging. Under the excitation wavelength of 320nm, the maximum emission wavelength of carbon dots (CDs) appears at 440nm and strong blue fluorescence is emitted. Methylene blue can be used for quenching the fluorescence of the carbon dots, and in a cell culture dish, a living cell incubates for 24 hours with the methylene blue, and the cell is observed by using a laser confocal microscopy system and continuously emits blue fluorescence. According to the preparation and the application, the carbon dots carbonized by semen litchi can be used for detecting the methylene blue by quenching a fluorescent signal. As an effective means for detecting the methylene blue, the method shows a remarkable advantage. The detection limit is as low as 0.05micro mol / L and the linear response range is 0.8-10micro mol / L [R<2>=0.9930]. As being a fluorescent probe, a sensing system has the optical characteristics of simplicity, low cost, greenness, high selectivity, rapidness and sensitivity and is successfully applied to methylene blue detecting and living cell imaging analysis. The sensing method has a broad application prospect in the field of environmental analysis and the cell imaging analysis.