Garlic is widely used as food flavouring, and China is the world's largest garlic producer and exporter. To develop a convenient technique for evaluation of garlic cultivars would be worthwhile, and it would have wide application in such a huge market. In this research, 3D front-face fluorescence data of 8 garlic cultivars were recorded, and independent component analysis was used to decompose the overall fluorescence spectra into six independent components. The first, second, and fourth independent components showed a big difference among the cultivars, and the chemical fluorophores behind these three components were specified as protein, vitamin B6, and ATP, respectively, as fluorescent markers for evaluation. The result showed that all 8 cultivars cluster separately. The cultivar “YNQJ” have the highest quality in terms of protein and vitamin B6, “NXYC” and “SDHB” have the highest content of ATP, while “SXXA” is the poorest in terms of protein and ATP, and “HNJZ” has the lowest content of vitamin B6. Therefore, rapid evaluation of garlic cultivars can be accomplished successfully by using only the proportion values of three properly selected fluorescent markers.