Carotenoid synthesis in Rhodotorula glutinis was investigated with Raman tweezers in order to find the effect of nitrogen and carbon resource on carotenoid yield.The cells in fermentation terminus were harvested, and then divided into two parts, one for UV analysis, the other for Raman tweezers detection.Original spectra were preprocessed by carrying out background elimination and baseline correction, and the averaged spectra of cells cultivated in different fermentation medium were analyzed qualitatively.The results showed that the Raman intensity of carotenoid were obviously different.There was a high correlation between UV results and Raman peak height data, the correlation coefficients of fitted parameters were 0.907 8 and 0.912 1, respectively.Quantitative analysis of 1 508 cm-1 peak height indicated that the appropriate nitrogen and carbon resources for the growth of Rhodotorula glutinis cells and synthesis of carotenoid were yeast extract+tryptone, and glucose, respectively.The above results suggest that Raman tweezers can provide information about carotenoids in Rhodotorula glutinis cells and serve as an effective tool for real time measurement of carotenoid synthesis and optimization of fermentation medium.