This paper studied the fluorescence spectral characteristic of chlorin e6-C15-monomethyl ester in different solvents to develop a fluorescence spectrometry for determining the concentration of chlorin e6-C15-monomethyl ester in plasma. By comparing the fluorescence spectral characteristics of chlorin e6-C15-monomethyl ester in six different solvents including methanol, ethanol, acetone, acetonitrile, phosphate buffered saline and water, the influence of different solvents on the fluorescence spectral characteristic of chlorin e6-C15-monomethyl ester has been examined. The results indicated that methanol and acetonitrile are the ideal solvent system, and the effect of phosphate buffered saline is better than water. The effect of organic solvent content and solution pH value on emission wavelength and fluorescence intensity was further evaluated. It was found that the fluorescence intensity of chlorin e6-C15-monomethyl ester was strong and stable in pH 7.2 phosphate buffered saline -acetonitrile (3∶7) solution and can be detected at the excitation wavelength of 498.00 nm and the emission wavelength of 664.05 nm. Based on this, we developed the fluorescence spectrometry for determining chlorin e6-C15-monomethyl ester. It is specific and sensitive with good linearity over the range of 0.5～50 μg·mL-1. The intra-batch and inter-batch precisions (RSD) were less than 10%, the extraction recoveries were all over 90%. The established method in this paper was simple, fast, effective and could be successfully applied to study the pharmacokinetics of chlorine e6-C15-monomethyl ester in SD rats.