Fluorescence spectroscopy with an excitation-emission matrix (EEM) is a fast and inexpensive technique and has been applied to the detection of a very wide range of analytes. However, serious scattering and overlapping signals hinder the applications of EEM spectra. In this contribution, the multi-resolution capability of Tchebichef moments was investigated in depth and applied to the analysis of two EEM data sets (data set 1 consisted of valine-tyrosine-valine, tryptophan-glycine and phenylalanine, and data set 2 included vitamin B1, vitamin B2 and vitamin B6) for the first time. By means of the Tchebichef moments with different orders, the different information in the EEM spectra can be represented. It is owing to this multi-resolution capability that the overlapping problem was solved, and the information of chemicals and scatterings were separated. The obtained results demonstrated that the Tchebichef moment method is very effective, which provides a promising tool for the analysis of EEM spectra. It is expected that the applications of Tchebichef moment method could be developed and extended in complex systems such as biological fluids, food, environment and others to deal with the practical problems (overlapped peaks, unknown interferences, baseline drifts, and so on) with other spectra.