Three-dimensional fluorescence spectroscopy was employed to investigate the fluorescence characteristic of chilled pork stored at different temperatures in this research. The locations of fluorescence peaks were identified and the changes in the average intensity values of two fluorescence peaks in their respective regions during storage were traced. Initially the fluorescent substances were determined as a basis for realizing rapid non-destructive detection of chilled pork freshness with three-dimensional fluorescence spectroscopy. The results showed that three-dimensional fluorescence spectra of chilled pork samples showedfluorescence peaks of two types (Peak A and Peak B) regardless of the storage temperature. The excitation wavelength (λex)/emission wavelength (λem) of Peak A was 250～310 nm/300～400 nm, whereas of Peak B was 300～450 nm/400～550 nm. Peak A and Peak B represented protein-like fluorescence and lipid oxidation products fluorescence, respectively. Moreover, the location of the maximum of Peak A was at λex/λem=290 nm/335 nm during storage, while that of Peak B shifted from λex/λem=320 nm/470 nm to λex/λem=390 nm/470 nm. The results also indicated that the average intensity values of two fluorescence peaks in their respective regions had the same trend regardless of the storage temperature: the average intensity values of Peak A in the region of λex/λem=250～310 nm/300～400 nm (IA) was gradually declined, while that of Peak B in the region of λex/λem=300~450 nm/400~550 nm (IB) was gradually increased as time went by. However, the storage temperature determined the change rate of IA and IB: the samples stored at 20 ℃ had higher the rate of change than those stored at 4 ℃.