Engineering three-dimensional (3D) hydrogels with well-defined architectures has become increasingly important for tissue engineering and basic research in biomaterials sci- ence. To fabricate 3D hydrogels with (sub)cellular-scale fea- tures, two-photon polymerization (2PP) shows great promise although the technique is limited by the selection of appropri- ate hydrogel precursors. In this study, we report the synthesis of gelatin hydrolysate vinyl esters (GH-VE) and its copolymer- ization with reduced derivatives of bovine serum albumin (acting as macrothiols). Photorheology of the thiol-ene copoly- merization shows a much more rapid onset of polymerization and a higher end modulus in reference to neat GH-VE. This allowed 2PP to provide well-defined and stable hydrogel microstructures. Efficiency of the radical-mediated thiol-vinyl ester photopolymerization allows high 2PP writing speed (as high as 50 mm s 21 ) with low laser power (as low as 20 mW). MTT assays indicate negligible cytotoxicities of the GH-VE macromers and of the thiol-ene hydrogel pellets. Osteosar- coma cells seeded onto GH-VE/BSA hydrogels with different macromer relative ratios showed a preference for hydrogels with higher percentage of GH-VE. This can be attributed both to a favorable modulus and preferable protein environment since gelatin favors cell adhesion and albumin incurs nonspe- cific binding. V C 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 4799-4810