Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic post-grafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to pre-formed PEG hydrogels containing the sortase LPRTG substrate. In addition, this technique was also adapted to functionalize 3D printed scaffolds. The simplicity, modularity and high specificity of sortase-mediated ligation makes it an attractive approach for modification of different biomaterials.