A capillary electrophoresis system with charge-coupled device multi-wavelength fluorescence detection was used for the separation and identification of fluorescence agents. Based on the differences between agents and impurities in fluorescence spectra and retention times shown in the three-dimensional electropherograms and by the use of a standard addition procedure, the fluorescence impurities in agents were identified. A novel method for the determination of the purity of fluorescence agents was proposed and applied to the purity analysis of three kinds of fluorescence agents.