A method based on molecularly imprinted solid-phase microextraction (MIP-SPME) coupled with liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) was developed for the detection of luteolin and its metabolites in vivo. The MIP-SPME fibers were first fabricated by dopamine and silane, and then luteolin MIPs-coated fibers were successfully prepared using luteolin, acrylamide (AM), and ethylene glycol dimethacrylate (EGDMA) as the template, functional monomer and cross-linker, respectively. The characterizations of polymers were analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and the Brunauer-Emmett-Teller method (BET). The properties involving adsorption and selective experiments were evaluated, and these results revealed that MIP fibers presented high adsorption capacity and selectivity to luteolin. Furthermore, the developed MIP-SPME coupled with the LC-QTOF-MS/MS method was adopted to capture and identify luteolin and its metabolites in rat livers in vivo, and eventually, apigenin, chrysoeriol, and diosmetin were rapidly identified as metabolites.