Compared with previous encapsulation methods, the emerging microfluidic approach has the potential of preparing monodispersed particles with considerable advantages, such as controlled size and morphology, stable yield, and enhanced repeatability. In addition to introducing probiotics to the intestine with high-viability, designing a novel carrier matrix to improve the mucoadhesion of probiotics in the intestine is imperative. Thus, in this study, the thiol groups of cysteine were grafted to chitosan to form disulfide bonds with the cysteine residues on the bacterial surface and mucus to achieve the dual function of probiotic adhesion and mucoadhesion. Upon examination, the adhesion rate of Bifidobacterium bifidum FL-276.1 (FL-276.1) to thiolated chitosan film increased by 1.33 times compared with non-modified chitosan film. Thiolated chitosan microspheres prepared by microfluidic method assisted by a crosslinking unit with a crosslinker of genipin had a uniform size distribution, high loading efficiency (80%) to FL-276.1, and improved resistance to simulated gastrointestinal digestion with a survival rate of FL-276.1 up to 92%. Moreover, the results of mucoadhesion test of thiolated chitosan microspheres demonstrated that the amount of thiolated chitosan microspheres adhered to the HT-29 cells was 2.27 times higher than that of chitosan microspheres. The monodispersed cysteine modified chitosan microspheres with dual adhesion function prepared by microfluidic technology has the potential to enhance the viability and function of probiotics after gastrointestinal digestion and intestinal adhesion.