<p>Meropenem (MEM), a broad-spectrum carbapenem antibiotic, has a narrow therapeutic window and high interindividual variability. Existing detection methods are often limited by complex operations, long turnaround times, and inadequate sensitivity. To overcome these challenges, this study developed a novel lateral flow immunochromatographic assay (LFIA) platform with synchronous triple-signal output for rapid and accurate MEM detection. A fluorescent membrane sensor was fabricated by integrating Rhodamine 6G (R6G) as a fluorescence donor into a nitrocellulose membrane. Isoniazid (INH, code 064) was selected as the optimal Raman reporter, enabling the synthesis of Raman nanospheres (R-Sphere⁰⁶⁴) capable of delivering colorimetric, surface-enhanced Raman scattering (SERS), and background fluorescence-quenched immunochromatographic assay (bFQICA) signals simultaneously. Density functional theory (DFT) calculations indicated that the adsorption of INH on the nanosphere surface involves an electron injection effect that promotes charge transfer, with an adsorption energy of -0.19&#xa0;eV and a charge transfer amount of 0.14 e. The characteristic SERS peak at 1006&#xa0;cm⁻¹ was attributed to the breathing vibration of the pyridine ring in INH. With a 15&#xa0;min detection time, the platform offers LODs of 0.8 ng mL^− 1 (SERS) and 0.8&#xa0;ng mL^− 1 (bFQICA), a shared LoQ of 1 ng mL^− 1, and a linear range of 1–10 ng mL^− 1. It demonstrated high stability, reproducibility, and specificity. Validation using 22 clinical samples showed strong correlation with standard clinical methods. The platform can be easily adapted to monitor other therapeutic drugs by replacing the specific antibody, offering a universal solution for clinical therapeutic drug monitoring (TDM).</p> Graphical Abstract <p></p>