In the human body, highly organized tissues rely on the compartmentalization effect of basement membranes (BMs) that separate different types of cells. We recently reported an artificial basement membrane (A-BM) composed of type-IV collagen and laminin (Col-IV/LM), which are the main components of natural BMs, for cell compartmentalization in three-dimensional (3D) tissues. However, such compartmentalized structures can be maintained only for 3 days, probably due to the degradation issues. In this study, a robust A-BM was fabricated by in situ cross-linking the Col-IV/LM layer-by-layer (LbL) nanofilms in 3D tissues by transglutaminase. The effects of molecular size and configuration on the permeability of obtained A-BMs were comprehensively studied using polystyrene nanoparticles (PS NPs) and dextran with various hydrodynamic diameters, as well as albumin. The findings agreed well with the known size-selective behavior of the glomerular basement membrane. Cross-linked Col-IV/LM nanofilms demonstrate improved stability and a more powerful barrier effect to maintain cell compartmentalization for organized 3D tissues. This in vitro A-BM exhibit great potentials for the design of more complex compartmentalized 3D tissues, for understanding the unique cell-cell cross talk through BMs, and for providing a more reliable 3D tissue model for new drug screening and other in vitro physiological studies.