We report the in situ generation of pH gradients in microfluidic devices for biofabrication of freestanding, semi-permeable chitosan membranes. The pH-stimuli-responsive polysaccharide chitosan was enlisted to form a freestanding hydrophilic membrane structure in microfluidic networks where pH gradients are generated at the converging interface between a slightly acidic chitosan solution and a slightly basic buffer solution. A simple and effective pumping strategy was devised to realize a stable flow interface thereby generating a stable, well-controlled and localized pH gradient. Chitosan molecules were deprotonated at the flow interface, causing gelation and solidification of a freestanding chitosan membrane from a nucleation point at the junction of two converging flow streams to an anchoring point where the two flow streams diverge to two output channels. The fabricated chitosan membranes were about 30-60 microm thick and uniform throughout the flow interface inside the microchannels. A T-shaped membrane formed by sequentially fabricating orthogonal membranes demonstrates flexibility of the assembly process. The membranes are permeable to aqueous solutions and are removed by mildly acidic solutions. Permeability tests suggested that the membrane pore size was a few nanometres, i.e., the size range of antibodies. Building on the widely reported use of chitosan as a soft interconnect for biological components and microfabricated devices and the broad applications of membrane functionalities in microsystems, we believe that the facile, rapid biofabrication of freestanding chitosan membranes can be applied to many biochemical, bioanalytical, biosensing applications and cellular studies.