The volatile organic compounds of six spices, including black pepper, dried ginger, cinnamon, fennel, clove, and zanthoxylum, were analyzed by gas chromatography-ion mobility spectrometry (HS-GC-IMS) combined with principal component analysis (PCA) and Euclidean distance. In further analyses, the effects of volatile oils in six spices on ulcerative colitis were assayed in a zebrafish model induced by 3-nitrobenzenesulfonic acid. A total of 120 kinds of volatile organic compounds were detected and 80 among them were identified, which included 10 common components and 3 to 24 characteristic components belonging to different spices. The major VOCs in six spices were estimated to be terpenes with the contents of 45.02%, 56.87%, 36.68%, 58.19%, 68.68%, and 30.62%, respectively. Meanwhile, the volatile components of fennel, dried ginger, black pepper, and cinnamon are quite similar, but differ from clove and zanthoxylum. The volatile oils in six spices presented efficient activity to improve ulcerative colitis which can decrease the number of neutrophils, restore the structure of intestinal epithelial and the morphology of the epithelial cells. Our study achieved rapid analysis of the volatile organic compounds and flavors in six spices and further revealed the potential health benefits of their volatile oils on ulcerative colitis, especially for clove and zanthoxylum. This study is expected to provide certain data support for the quality evaluation and the potential use in functional foods of six spices.