The work aims to prepare a screen printing ink with excellent anti-counterfeiting effect, printability, and environmental protection by optimizing the synthesis method of carbon dots and the ink formulation. Firstly, with o-phenylenediamine as a carbon source and water and ethanol as solvents, red and yellow carbon dots were prepared by the solvent (hydro) thermal method. Blue carbon dots were prepared with sodium citrate and ammonium bicarbonate as carbon and nitrogen sources. The structural compositions and optical properties of the three carbon dots were characterized and analyzed. Then, tricolor fluorescent carbon dots were used as pigments, ethanol or water was selected as the solvent, and water-based epoxy resin and polyacrylic resin were used as binders to obtain the optimum proportion through test to prepare tricolor fluorescent anti-counterfeiting ink. Through the test, it was found that the tricolor carbon dots had relatively uniform sizes. Under the excitation of 365 nm ultraviolet light, they emitted red fluorescence at 725 nm, blue fluorescence at 450 nm and yellow fluorescence at 570 nm, and the fluorescence quantum yields of rCDs, bCDs and yCDs were 56.63%, 64.37%, and 78.26%, respectively. By optimizing the synthesis method of carbon dots, the fluorescence emission spectrum can be controllably adjusted, and tricolor carbon dots with wide ultraviolet absorption band, narrow emission spectrum band and high fluorescence quantum yield can be prepared. This carbon dot can be used as fluorescent pigment to prepare water-based ink with good printability. It meets the requirements of fluorescent anti-counterfeiting printing.