Pigments are important constituents in tobacco, that relate to appearance and quality of tobacco, meanwhile, the degradation products of pigments have great effects on the quality of aroma of tobacco. According to their color and property, pigments aregenerally divided into three categories: green pigment, yellow pigment and melanin. In growth, chlorophyll is the main green pigment in tobacco, while xanthophyll and carotene are the main yellow pigments in mature tobacco. Melanin consists in the progress of modulation and fermentation when tobacco leaves are ripe. Analysis of pigments in tobacco is important for evaluations of raw tobacco and the tobacco product quality. Traditional analytical methods of pigments in tobacco based on liquid chromatography, normally need long time and complex sample preparation process, while Raman spectroscopic method is simple to operate and takes short determination time, so that it could be able to provide information about molecular functional groups. Accordingly a method of simultaneous detection of lutein and β-carotene in tobacco was developed with Raman spectroscopy in the present work. A solution of organic solvent extraction from a tobacco sample was enclosed in a glass vial and was detected to collect its Raman spectrum by focusing the laser on the solution inside. The excitation light wavelength was optimized and 455 nm was selected, under which high Raman signals were obtained. Other experiment conditions, including extracting solvent of pigments and the distance between the focal plane and the optical platform were optimized. Because the spectra were measured in different dates and the measurement conditions could change, normalization was used to correctphysical interference due to the changes with a selected internal standard peak from the solvent spectrum. To solve problem of spectroscopic interference due to severe overlap between Raman signals of lutein and β-carotene, partial least squares (PLS) was utilized to build multivariate calibration models between Raman intensities and concentrations of pigments. The results showed that the normalization process could help to correct the changes of the measurement conditions, derivative calculations cannot improve the models but the models can be improved significantly by selection of wavelength regions. When spectral regionswere selected between 798.2 and 1 752.8 cm-1 for lutein, regions of 798.2～1 752.8 and 2 254.2～2 784.5 cm-1 for β-carotene, the built PLS models showed the best performances with the prediction errors of RMSEP being 6.68 and 2.56 μg·g-1, respectively. The results indicated that Raman spectra combined with PLS could supply a new way to the quantitative analysis of lutein and β-carotene in tobacco with advantages of easy operation, short time and reliable prediction.