The work aims to better study the imaging mechanism of the additive color method, improve the fluorescence performance of the red fluorescent screen printing ink, and then study the coloration performance of the three-color fluorescent ink. The fluorescence performance of red water-based fluorescent screen printing ink was studied by changing the content of red fluorescent material. Based on that, the color gamut of red, green and blue fluorescent ink was obtained. The effects of different coloration methods on the fluorescence performance of three-color ink were compared and the effects of two different substrates on the coloration performance of three-color ink were investigated. When the phosphor content was 6%, the fluorescence intensity of red water-based fluorescent screen printing ink was the best. The color gamut range of three-color fluorescent ink was smaller than sRGB, but it could meet the needs of color reproduction research. For screen printing, compared with dot superposition, the dot coordination was more favorable to the true representation of fluorescent ink color information. The substrate containing no fluorescent agent was in favor of color reproduction of the fluorescent ink. The paper was better than PC film in hue reproduction ability. The water-based fluorescent screen printing ink finally prepared has excellent fluorescence performance and can reproduce most of the colors in sRGB color gamut.