Microscale fabrication of three-dimensional (3D) extracellular matrices (ECMs) can be used to mimic the often inhomogeneous and anisotropic properties of native tissues(1-3) and to construct in vitro cellular microenvironments(4-6). Cellular contraction of fibrous natural ECMs (such as fibrin and collagen I) can detach matrices from their surroundings and destroy intended geometry(7-9). 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.