Microscale fabrication of three-dimensional (3D) extracellular matrices (ECMs) can be used to mimic the often inhomogeneous and anisotropic properties of native tissues and to construct in vitro cellular microenvironments. Cellular contraction of fibrous natural ECMs (such as fibrin and collagen I) can detach matrices from their surroundings and destroy intended geometry. Here, we demonstrate in situ collagen fibre assembly (the nucleation and growth of new collagen fibres from preformed collagen fibres at an interface) to anchor together multiple phases of cell-seeded 3D hydrogel-based matrices against cellular contractile forces. We apply this technique to stably interface multiple microfabricated 3D natural matrices (containing collagen I, Matrigel, fibrin or alginate); each phase can be seeded with cells and designed to permit cell spreading. With collagen-fibre-mediated interfacing, microfabricated 3D matrices maintain stable interfaces (the individual phases do not separate from each other) over long-term culture (at least 3 weeks) and support spatially restricted development of multicellular structures within designed patterns. The technique enables construction of well-defined and stable patterns of a variety of 3D ECMs formed by diverse mechanisms (including temperature-, ion- and enzyme-mediated crosslinking), and presents a simple approach to interface multiple 3D matrices for biological studies and tissue engineering.