<p>Tacrolimus (Tac), a macrolide immunosuppressant widely employed in organ transplantation, requires therapeutic drug monitoring owing to the narrow treatment window. In this study, we established an innovative ultrabright nanoparticle-based lateral flow immunoassay (LFIA) for one-step rapid quantitative detection of Tac in human plasma. The aggregation-induced emission (AIE) luminogen, AIE490, was embedded in polystyrene nanoparticles to assemble the ultrabright AIE490NP as a highly sensitive fluorescence marker. Carboxyl-modified Tac was used to couple with carrier proteins to synthesize complete antigens coated on the test line. Under optimized conditions, the limit of detection for Tac is 0.589 ng/mL in human plasma. The linear range was 1–1000 ng/mL, completely covering the safe plasma concentration of Tac, 5–15 ng/mL. The intra- and inter-assay precisions of the established method are below 17.99% and 19.94%, respectively. In addition, for measuring the plasma concentration of Tac in 139 clinical samples, the results of the LFIA are consistent with those of LC–MS/MS (<i>R</i>² = 0.8520, <i>P</i> &lt; 0.0001). Therefore, the established LFIA demonstrates comparable reliability to the LC–MS/MS and holds great potential as a rapid quantitative detection method of Tac in human plasma.</p> Graphical abstract <p></p>