The invention discloses a multi-color fluorescent carbon quantum dot and a preparation method and application thereof, and relates to a multi-color fluorescent carbon quantum dot and a preparation method thereof. The preparation method comprises the following steps of: putting ammonium citrate and urea into a reaction container, adding dimethylformamide, fully stirring to obtain a transparent clear solution, transferring the clear solution into a high-pressure kettle, putting the high-pressure kettle into a drying oven, and keeping the temperature at 150-200 DEG C for 14 hours to obtain a carbon quantum dot solution. The carbon quantum dot prepared by the invention has the advantages of controllable light emitting color, excellent optical performance, strong fluorescence, high quantum yield, stability, small influence of acid-base environment and the like. The raw materials of the multi-color carbon quantum dot prepared by the method are cheap and easily available, the experimental repeatability is high, the operation is simple, the preparation time is short, and large-scale production can be realized. The multi-color fluorescent carbon quantum dot is applied to the fields of bioluminescence labeling, white light illumination and the like.