The integration of membranes in microfluidic devices has been extensively exploited for various chemical engineering and bioengineering applications over the past few decades. To augment the applicability of membrane-integrated microfluidic platforms for biomedical and tissue engineering studies, a biologically friendly fabrication process with naturally occurring materials is highly desired. The in situ preparation of membranes involving interfacial reactions between parallel laminar flows in microfluidic networks, known as the flow-assembly technique, is one of the most biocompatible approaches. Membranes of many types with flexible geometries have been successfully assembled inside complex microchannels using this facile and versatile flow-assembly approach. Chitosan is a naturally abundant polysaccharide known for its pronounced biocompatibility, biodegradability, good mechanical stability, ease of modification and processing, and film-forming ability under near-physiological conditions. Chitosan membranes assembled by flows in microfluidics are freestanding, robust, semipermeable, and well-aligned in microstructure, and show high affinity to bioactive reagents and biological components (e.g. biomolecules, nanoparticles, or cells) that provide facile biological functionalization of microdevices. Here, we discuss the recent developments and optimizations in the flow-assembly of chitosan membranes and chitosan-based membranes in microfluidics. Furthermore, we recapitulate the applications of the chitosan membrane-integrated microfluidic platforms dedicated to biology, biochemistry, and drug release fields, and envision the future developments of this important platform with versatile functions.