The invention discloses a preparation and a fluorescence regulation method of water-soluble non-toxic carbon quantum dots. The method comprises the following steps: 1) preparing a carbon quantum dot precursor solution; 2) preparing carbon quantum dots: transferring the precursor solution into a reaction kettle, performing a reaction at 200 DEG C for 24 h, performing natural cooling after reaction,and performing purification by a dialysis bag to obtain the carbon quantum dots; and 3) performing a fluorescence regulation process: regulating the B and N doped carbon quantum dots to emit light byutilizing the pH value and the filling degree of the reaction kettle to prepare the carbon quantum dots with different fluorescence wavelengths and different light emitting intensities. The method has the advantages of simplicity in operation, and low operation process difficulty, and the fluorescence wavelength of the obtained carbon quantum dots does not change along with the change of excitinglight energy.