Herein, for the first time, thiol-functionalized mesoporous silica (mSiO(2)-SH) nanofibers with a spirally-curved twisted hexagonal morphology were synthesized via a simple one-pot protocol. 4-Mercaptophenylboronic acids (4-MPBA) were attached onto the mSiO(2)-SH nanofibers via disulfide bond, serving as boronate affinity sorbent to selectively capture brassinosteroids (BRs) from plant extract. The resulting BRs-MPBA derivatives were easily eluted from the sorbent by cleaving the disulfide bond, which was subsequently subjected to ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Thus, the in situ extraction/derivatization/desorption method coupled with UPLC-MS was established for the fast, sensitive and selective detection of BRs in plant tissues. Finally, based on the developed method, endogenous BRs were successfully detected in leaf of H. lupulus L., silique of A. thaliana, and panicle of O. sativa L.