A method is established based on laser tweezer Raman spectroscopy (LTRS) for rapid quantitation of lipid and carotenoids in Rhodosporidium toruloides cells, and the effect of different C/N, C/P, C/S ratios in a culture medium on synthesis capability of lipid and carotenoids is evaluated at the single-cell level. The Raman spectra of plant oil in different concentrations are determined and the high correlation between the peak intensity at 1751 cm-1 and lipid concentration is observed. The correlation coefficient of standard curve for lipid is 0.97349. The data demonstrate that LTRS is efficient in the quantitative analysis of lipid. Lipid and carotenoids within the Rhodosporidium toruloides cells cultured in different media are acquired quantificationally according to the Raman peak heights at 1751 cm-1 and 1520 cm-1, respectively. The result shows that the lipid concentration in Rhodosporidium toruloides cells increases as the ratios of C/N, C/P, C/S increase, while the carotenoids concentration significantly decreases. The above results indicate that LTRS is an efficient approach to the quantitative analysis of lipid and carotenoids in microbial cells and the optimization of fermentation medium.