Probiotics play a crucial role in regulating intestinal immune homeostasis and supporting gut health; however, oral administration faces challenges such as nonspecific distribution and low efficacy. To achieve precise and efficient delivery, in this study, a targeted delivery system embedded probiotics to the colonic inflammatory site CD44 was constructed by covalently linking hyaluronic acid (HA) to cysteine-modified chitosan (CCH) using microfluidic technology. Two probiotic strains, Bifidobacterium bifidum FL-276.1 and Clostridium butyricum ATCC 19398, were encapsulated within the modified chitosan matrix to form probiotics@CCH microspheres (MSs), with an average diameter of approximately 276 mum. Based on the receptor-ligand binding mechanism of HA and CD44, combined with the intestinal mucosal adhesion properties conferred by cysteine-modified chitosan, the probiotics@CCH MSs exhibited a high capture rate for inflammatory Caco-2 cells and demonstrated prolonged retention and targeted localization in a DSS-induced colitis mouse model. Furthermore, probiotics@CCH MSs contributed to maintaining intestinal homeostasis by modulating gut microbiota composition, enhancing short-chain fatty acid production, and supporting the intestinal barrier integrity. The microfluidic-based delivery system facilitates the precise localization of probiotics within the intestine, providing a theoretical basis for enhancing probiotic applications in gut health management.