A method based on on-line solid-phase extraction (SPE) with hydrophobic monolithic column coupled with high performance liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of five avermectins in bovine liver. A poly(butyl methacrylate-co-ethylene dimethacrylate) monolithic column was used as the sorbent. The parameters influenced on on-line SPE and separation process such as the loading mobile phase, the eluting flow rate and the solvent for the separation were investigated in detail. Blank samples, spiked samples, matrix effect and recovery experiments were investigated to evaluate the extraction efficiency and potential interfering compounds originating from the matrix. Under the optimized conditions, the method showed a linear range of 1-100 µg/L and the quantification limit of 5 µg/kg for each analyte. The presented method gave recoveries of 77.4%-98.4%. The relative standard deviations of intra-day and inter-day were 4.46%-8.03% and 4.79%-8.68%, respectively. Moreover, no significant changes were found in the extraction performance after more than 400 usages on one monolithic column, and even on the monoliths with various batches. The feasibility of the developed poly (butyl methacrylate-coethylene dimethacrylate) monolithic column based on the on-line SPE method for the determination of avermectins was further demonstrated by the analysis of real samples.