The present study investigated the three-dimensional spectra and emission spectra of the autofluorescence of rabbit hearts. The results suggested that the three-dimensional spectra of the iced atria and ventricle were observed more evidently different from that of the fresh tissue compared to the main artery, which indicated that the amount of flavins and NADHs changed. Also, the atria, ventricle and main artery have different specific excitation spectra at the wavelength of 340 nm. The main fluorescence peaks were of NADH (at about 460 nm), collagen and elastin (at about 290-400 nm).The Gauss spectra of atria and ventricle were different in the peak value, relative intensity and half width. So the ratios of fluorescence intensities of peaks may be used to distinguish different heart tissues. Furthermore, a phenomenon was firstly uncovered that the autofluorescence intensity of NADH in ventricle decays with the time of death and it could be a useful method for the estimation of postmortem interval.