We report an in situ biofabrication strategy that conveniently partitions microfluidic networks into physically separated while chemically communicating microchannels with semipermeable biopolymer membranes, which enable the facile generation of static gradients for biomedical applications. The biofabrication of parallel biopolymer membranes was initiated with the dissipation of trapped air bubbles in parallel apertures in polydimethylsiloxane (PDMS) microfluidic devices, followed by tunable membrane growth with precise temporal and spatial control to the desired thickness. Static gradients were generated within minutes and well maintained over time by pure diffusion of molecules through the biofabricated semipermeable membranes. As an example application, the static gradient of alpha factor was generated to study the development of the "shmoo" morphology of yeast over time. The in situ biofabrication provides a simple approach to generate static gradients and an ideal platform for biological applications where flow-free static gradients are indispensable.