Sophora tonkinensisis widely used as traditional Chinese medicine for treating the swelling of the gums and tongue and mouth sores due to flame stomach fire. It is mainly origin from Guangxi, Sichuan provinces of China. Alkaloids are considered as the major bioactive components. A method was established for identifying alkaloids inS. tonkinensisroot by UPLC-Q-TOF-MS/MS and was applied in characterizing alkaloids inS. tonkinensisroot of two different habitats. Consequently, twenty-four alkaloids including six new compounds were identified inS. tonkinensisroot. Additionally, the difference of alkaloids inS. tonkinensisfrom Guozhou, Sichuan province was investigated. In the present study, we firstly characterize total alkaloids inS. tonkinensisroot by UPLC-Q-TOF-MS/MS and firstly established the characteristic fragmentation pathway of alkaloids with hydroxy inS. tonkinensisroot.