Vancomycin (VAN) has a narrow therapeutic window and variable pharmacokinetic properties, which require timely monitoring of plasma concentration to adjust the dosage for better effectiveness. A sensitive and quantitative immunochromatographic method was developed to detect VAN in plasma. The linear response range of the method to detect plasma VAN was 1.25–50 μg/mL. The intra‐ and inter‐assay coefficients were 4.68% and 8.81%, respectively, while the recovery was 89.10%–98.32%. The clinical comparative experiment results demonstrated a good correlation (r > 0.90) between the fluorescent immunochromatographic strip test and the mass spectrum. In this study, a simple, rapid, and high‐sensitivity immunochromatographic method was established for detecting VAN, which helped establish the basis for further application of monitoring plasma concentration.