A rapid and simple method with online solid phase extraction (SPE) has been developed for the simultaneous determination of beta-lactam antibiotics (BLAs) (amoxicillin, cephradine, and cefazolin sodium) in aquatic environment and milk. The epoxy-based organic–inorganic hybrid monolithic column was used as SPE sorbent to simultaneously monitor three analytes. The morphology of monolithic column and pressure drop across the columns were characterized. Excellent permeability and high selectivity were obtained. The linear range of the standard curve was from 2.0 to 500ng/mL (r 2 ≥0.999). Precision for inter- and intra-day assay showed acceptable results for quantitative assay with relative standard deviation (RSD) less than 11%. The accuracy and recovery were found to be within the range of 93–103% and 83–105%. The results indicated that the prepared monolithic column could provide excellent reproducibility and implied that the prepared monolith was feasible to be used as an on-line SPE sorbent material.