Astaxanthin is a carotenoid widely used in food additives, nutritional product and medicines, which shows many physiological functions such as antioxidant, anti-inflammatory, anti-hypertensive and anti-diabetic activities. It has been recognized that astaxanthin has all-trans and nine cis isomers, and these geometrical isomers have very different biological activities. The process of selective enrichment, metabolism and isomerization of astaxanthin in animals remains to be studied. Therefore, identifying isomers and obtaining their structural parameters are important for understanding the active mechanism of different molecular isomers. Although the traditional methods such as high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy can be used to distinguish these isomers, these methods generally require considerable testing time, cost, sample volume, and hardly be applied in vivo. In this work, Raman spectroscopy combined with density functional theory (DFT) calculation was introduced to study different geometrical isomers of astaxanthin. The theoretical and experimental Raman spectra are in agreement, and we have demonstrated that all the known ten geometrical isomers of astaxanthin can be readily distinguished using this spectroscopic approach. The astaxanthin molecular vibrational modes, geometric structures, energies of ten geometric isomers are systematically scrutinized. Moreover, a lot of structural and Raman problems unsolved previously have been solved by the DFT-based spectral analysis. Therefore, this work provides an effective way for identification of different astaxanthin geometrical isomers, and may have important significance for promoting the research of astaxanthin isomers on biological property mechanisms and related applications in food molecular science.