This study investigated the absorption spectrum and the fluorescence spectrum of rabbit hearts stained with the voltage sensitive dye (di-4-ANEPPS). The results suggested that the optical absorption of tissue with the dye was higher than that of the control group, and there were significant differences between the experimental group and control group in the range of 450-550 nm. It was also indicated that the maximum absorption peak of the dye in tissues was at (479.25 +/- 0.44) nm. Additionally, the different peaks of fluorescence emission spectra from the atriums, ventricles and aorta were originally found by testing the five parts of rabbit hearts with the dye. Their relative intensities were related to the distribution concentrations of the dye. Meanwhile, the peaks of excitation spectra and fluorescence emission spectra were determined by examining the three-dimensional and two-dimensional fluorescence spectra using ventricles and atriums with the dye. Based on the discrepancy of rest membrane voltages between ventricles and atriums, the best wavelength ranges of excitation light and emissions light of optical mapping were determined by the shifts of the dye in emission spectra with excitation at different wavelengths. These results offer a theoretical foundation for the design of a cardiac optical mapping system.