Abstract: Fermented traditional Chinese medicines (TCMs) dated to classic age and its modern research develops more in-depth. Fermentation plays an important role in toxicity reducing and efficacy enhancing for TCMs, but its material basis is inadequate. Hibiscus sabdariffa L. is a homology of medicine and food plant that has a good development of medicinal and utilization of value with its rich nutrient. It has too much content of phenolic acid to taste sour and hard to accept. Studies have shown that fermentation broth from Hibiscus sabdariffa L. developed a nutrient-rich, sweet and sour and refreshing, unique flavor, with good stability product. In order to demonstrate which constituent has been changed during fermentation, ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) equipped with an ESI ion source at negative mode was used for comprehensive study of the chemical constituents of aqueous extracts and fermentation broth from Hibiscus sabdariffa L.. Separation was performed with an Agilent Zorbax SB C18 column (250 mm×4.6 mm×5 μm) with 0.1% formic aqueous solution and methanol as the mobile phase with the speed of 1 mL/min under the gradient condition. Based on the accurate mass measurement (error<5×10^-6), MS/MS fragmentation pathways, different chromatographic behaviors and compared with the standards and references, a total of 41 compounds, including 34 organic acids, 4 flavonoids, 2 anthocyanins and 1 lignan are tentatively identified and characterized. The results showed that the varieties and concentration of Hibiscus sabdariffa L. change in different degrees and they provide the scientific basis for the statement for Hibiscus sabdariffa L. and the changes of its chemical constituents after fermentation.