<p>Antibiotics are typically administered via direct injection into the body. However, maintaining a stable drug concentration is challenging, leading to reduced bioavailability and potential toxic side effects due to improper concentration control. To address this issue, this study designed a controlled antibiotic release system based on thiolated beta-cyclodextrin crosslinked complexes, using levofloxacin as a model drug. First, beta-cyclodextrin was thiolated and then crosslinked via click reactions between thiol and alkene groups. The resulting crosslinked beta-cyclodextrin efficiently encapsulated levofloxacin through its hydrophobic structure. This system is regulated by a specific physiological stimulus—glutathione (GSH)—which triggers the dynamic disulfide-thiol exchange, leading to the dissociation of the crosslinked structure of beta-cyclodextrin and the controlled release of levofloxacin, enabling on-demand drug delivery. The formation of crosslinked beta-cyclodextrin was characterized using NMR, FTIR, SEM and DLS. Drug release experiments demonstrated that the crosslinked beta-cyclodextrin exhibited excellent GSH-responsive release characteristics. Cytotoxicity assays confirmed the good biocompatibility of the system, while antibacterial tests further validated that GSH stimulation significantly enhanced the antibacterial efficacy of levofloxacin-loaded crosslinked beta-cyclodextrin. This study developed GSH-responsive crosslinked beta-cyclodextrin drug carrier, providing a novel strategy for the controlled release of antibiotics, with the potential to enhance therapeutic efficacy and achieve on-demand drug administration.</p>