By on-line solid phase microextraction with polymer monolith coupled to liquid chromatography-mass spectrometry (SPME-LC-MS), an automated and sensitive method for analysis of the endogenous 28-epihomobrassinolide (28-epihomoBR) in Arabidopsis thaliana was developed in this work. Firstly, A poly(methacrylic acid-co-ethylene dimethacrylate) (poly(MAA-co-EDMA)) monolith was prepared in the capillary and applied in in-tube SPME. Polyethylene glycol (PEG) was used as porogen to adjust the specific surface area and hydrophobicity of the target monolith to get satisfactory permeability, high mechanical strength and good stability. The optimized monolith was then served as extraction medium for analysis of the derivatized 28-epihomoBR in plant samples with the cleanup of matrix and enrichment of desired analyte at the same time. Good linearity was obtained in the linear range of 5-500 ng/L with the coefficient of determination (R2) of 0.9996. The limit of detection (S/N=3) of 28-epihomoBR was found to be 2.0 ng/L and the limit of quantification (S/N=10) was 5.0 ng/L. Using this method, the endogenous 0.101 ng/g (FW) 28-epihomoBR was successfully detected from only 400 mg Arabidopsis thaliana samples with satisfactory recovery (80.3-92.1%) and reproducibility (RSD 6.8-9.6%). Comparing with other sample pretreatment methods, this automated on-line SPME-LC-MS method is rapid, sensitive, reproducible and laborsaving.