Abstract: Water-extracted polyphenols in dandelion have been reported to possess a high potential for inhibit urease. Dandelion powder with whole grass was used to screen the main water-extracted polyphenols in dandelion with inhibiting urease, and study the inhibitory effects and mechanisms by HPLC-MS, Berthelot Method, Lineweaver-Burk method, fluorescence analysis and molecular docking in this paper. The results showed that main components of water-extracted polyphenols in dandelion were cichoric acid and caffeic acid, the contents were 10.62% and 7.39%, binding energies were −5.65 and −3.60 kcal/mol, and the sum of their inhibitory rates to urease accounted for 75.76% of the total water-extracted polyphenols in dandelion, indicating they are the major components to inhibit urease. IC₅₀ were 0.34±0.07 and 3.04±0.68 mmol/L, respectively. The inhibitory effects were both concentration-dependent and highly effective at low concentrations. They had the same action site against urease, the inhibition types were non-competitive inhibition type dominated by hydrogen bond and van der Waals force, and the fluorescence quenching mechanism is static quenching. This study would provide a theoretical basis for elucidating the main polyphenols of dandelion and their mechanism against urease, and promoting the development of polyphenol as urease inhibitors.