A method is established based on micro-Raman spectroscopy in situ quantitative detection technology for the screening of carotenoid high-producing mutants from rhodotorula glutinis colonies growing on solid culture medium. In order to eliminate the interference of medium background signal with the quantitative results, vector correction algorithm is used to preprocess spectral data to obtain pure bacterial signal without medium interference. The effect of two different spectral collection modes on spectral repeatability is investigated. The results show that spectral signal collected with area-scan excitation mode can possess better repeatability than with fixed-point detection. Confocal micro-Raman spectroscopy is applied to estimate carotenoid content of rhodotorula glutinis colonies on solid culture medium, which is verified by laser tweezers Raman spectroscopy. A good correlation between carotenoid Raman peak intensities at 1512 cm-1 by above two approaches is observed. The data indicate that confocal micro-Raman spectroscopy is a reliable approach for the quantitative analysis of carotenoids in rhodotorula glutinis colonies on solid culture medium. Two carotenoid high-producing variants are obtained with the screening procedure established above. This evidence confirms that the proposed strains screening method is more objective and efficient than conventional methods.