Spatial patterning of proteins (extracellular matrix, ECM) for living cells on polyacrylamide (PA) hydrogels has been technically challenging due to the compliant nature of the hydrogels and their aqueous environment. A traditional micro-fabrication process is not applicable. Here we report a simple, novel and general method to pattern a variety of commonly used cell adhesion molecules, i.e. fibronectin (FN), laminin (LN), collagen I (CN), etc. on PA gels. The pattern is first printed on a hydrophilic glass using a polydimethylsiloxane (PDMS) stamp and micro-contact printing (μCP). A pre-polymerization solution is applied on the patterned glass and is then sandwiched by a functionalized glass slide, which covalently binds to the gel. The hydrophilic glass slide is then peeled off from the gel when the protein patterns detach from the glass, but remain intact with the gel. The pattern is thus transferred to the gel. The mechanism of pattern transfer is studied in light of interfacial mechanics. It is found that hydrophilic glass offers a strong enough adhesion with ECM proteins such that a pattern can be printed, but a weak enough adhesion such that they can be completely peeled off by the polymerized gel. This balance is essential for a successful pattern transfer. As a demonstration, lines of FN, LN and CN with widths varying from 5–400 μm are patterned on PA gels. Normal fibroblasts (MKF) are cultured on the gel surfaces. The cell attachment and proliferation are confined within these patterns. The method avoids the use of any toxic chemistry often used to pattern different proteins on gel surfaces.