Chitosan-based biomaterials has shown great advantages in a broad range of applications, including drug delivery, clinical diagnosis, cell culture and tissue engineering. However, due to the lack of control over the fabrication processes by conventional techniques, the wide application of chitosan-based biomaterials has been hampered. Recently, microfluidics has been demonstrated as one of the most promising platforms to fabricate high-performance chitosan-based multifunctional materials with monodisperse size distribution and accurately controlled morphology and microstructures, which show great promising for biomedical applications. Here, we review recent progress of the fabrication of chitosan-based biomaterials with different structures and integrated functions by microfluidic technology. A comprehensive and in-depth depiction of critical microfluidic formation mechanism and process of various chitosan-based materials are first interpreted, with particular descriptions about the microfluidic-mediated control over the morphology and microstructures. Afterwards, recently emerging representative applications of chitosan-based multifunctional materials in various fields, are systematically summarized. Finally, the conclusions and perspectives on further advancing the microfluidic-aided chitosan-based multifunctional materials toward potential and versatile development for fundamental researches and biomedicine are proposed.