: Ultraviolet and visible radiations are absorbed by fluorescent dyes at short wavelengths and re-emitted at longer wavelengths. Total radiance factor of fluorescent dyes consists of true and fluorescent reflection. The common Kubelka-Munk color model describes the reflectance of a sample without considering fluorescence component; so, true spectral reflection of fluorescent materials should be separated from fluorescence emission. To this aim, there are some methods such as filter application to reduce fluorescence component, polychromatic and monochromatic spectrophotometers and computational methods to eliminate or decrease the fluorescence effect. In present study, to separate the true reflectance component from the fluorescence emission, the total spectral radiance factor of the fluorescent dyes was compared with the reflectance of the substrate through all visible wavelengths and exciting, crossover and emission regions were determined. Accordingly, the total radiance factor curve can be separated into two parts of true and fluorescence reflection considering the Kubelka–Munk theory for the true reflectance part. In this study, a simple and feasible method for color matching of fluorescent dyes have been developed. Two sets of equations to relate the fluorescence reflection and dye concentration were considered for single and mixture shades of fluorescent dyes. The amounts of dyes were determined by a correction matrix, which can minimize color difference values between target and prediction samples. The performance of the proposed method was evaluated by the color difference values, the root mean square differences of reflectance curves and the relative error of concentration prediction. This method is suitable for quantitative analysis of samples dyed with two fluorescent dyes.