The invention provides a preparation method of nitrogen-doped graphene quantum dot dispersion liquid. The preparation method comprises the following steps: placing a glucose-ammonium water mixed solution in a microwave-resisting non-sealed container, and carrying out the microwave radiation reaction; stirring the liquid obtained in the reaction at a low temperature, heating to remove excessive ammonium water, placing the liquid into a dialysis bag, centrifuging to accelerate the dialysis purification to obtain the nitrogen-doped graphene quantum dot dispersion liquid. According to the preparation method, a one-step synthetic method is adopted, the glucose is used as a carbon source, ammonium water is used as a nitrogen source and a drive agent, the microwave radiation is used as assistance, and the synthetic rate from the polymerization of glucose, nitrogen doping, carbonization to the formation of nitrogen-doped graphene quantum dots is extremely increased; the method is simple in process, easy for obtaining raw materials, rapid in reaction and environment-friendly; the prepared nitrogen-doped graphene quantum dot is good in monodispersity, high and adjustable in photoluminescent properties, excellent in solubility, good in application prospect in the fields of biological imaging, fluorescent probes and the like, and suitable for industrialized mass production after being amplified in the pilot test.