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β‐Blockers and β2‐agonists are commonly prescribed for therapeutic treatments and are also administered to livestock, leading to their presence in both environmental and biological samples. Hence, the development of sensitive, rapid, and reliable analytical methods for the determination of β‐blockers and β2‐agonists in environmental and biological samples is important. In this study, MIL‐101(Cr)‐NH2&GO‐coated SiO2/Fe3O4 magnetic particles were prepared as sorbents for magnetic solid‐phase extraction and then combined with high‐performance liquid chromatography‐tandem mass spectrometry for the analysis of 20 β‐blockers and eight β2‐agonists. The experimental parameters of magnetic solid‐phase extraction were studied in detail, and the optimal conditions were established. Under optimal conditions, the limits of detection were in the range of 0.002–0.007 μg/L with enrichment factors of 20.2–24.9. The developed method was successfully applied for the determination of 20 β‐blockers and eight β2‐agonists in river water, human urine, and freeze‐dried pork liver powder. Bisoprolol and salbutamol were detected at concentrations of 2.78 mg/L in human urine and 11.5 μg/kg in freeze‐dried pork liver powder. [ABSTRACT FROM AUTHOR]