•A MIP column was prepared by packing polymyxin E-imprinted microspheres.•MIP column showed excellent stability, efficiency and selectivity to polypeptide antibiotics.•In-line MISPE allowed the screening of polypeptide antibiotics in animal tissue by LC-UV.•A LC-MS/MS method based on in-line MISPE for the analysis of polypeptide antibiotics was developed.•Obtained recoveries ranged from 66.7 to 94.5% and limits of detection were 3.0 ∼ 8.0 μg/kg. Effective purification and enrichment of polypeptide antibiotics in animal tissues is always a challenge, due to the co-extraction of other endogenous peptides which usually interfere their final determination. In this study, a molecularly imprinted column was prepared by packing polymyxin E-imprinted particles into a 100 mm × 4.6 mm i.d. HPLC column. The as-prepared imprinted columns were able to tolerate 100% aqueous phase and exhibited good stability and high column efficiency. Polypeptides antibiotics with similar molecular size or spatial structure to polymyxin E were well retained by the imprinted column, suggesting class selectivity. After optimization of mobile phase conditions of imprinted column, polypeptide antibiotics in animal tissue extracts were enriched and cleaned up by in-line molecularly imprinted solid-phase extraction, allowing the screening of target analytes in complex samples at low concentration levels by UV detection. Eluate fraction from the imprinted column was collected, and further dried and re-dissolved with methanol-0.5% formic acid aqueous solution (80:20, v/v) for final LC-MS/MS analysis. Analysis was accomplished using multiple reaction monitoring (MRM) in positive electrospray ionization mode and analytes quantified using the matrix-matched external calibration curves. The results showed high correlation coefficients for target analytes in the linear range of 2 ∼ 200 μg kg−1. At four different concentration levels (limit of quantification, 50, 100 and 200 μg kg−1), recoveries of four polypeptide antibiotics in swine, cattle and chicken muscles ranged from 66.7 to 94.5% with relative standard deviations lower than 16.0%. The limits of detection (LOD) were 2.0 ∼ 4.0 μg/kg, depending upon the analyte and sample. Compared with a conventional pretreatment method, the imprinted column was able to remove more impurities and to significantly reduce matrix effects, allowing the accurate analysis of polypeptide antibiotics.