In this work, we present a method to fabricate a hyaluronic acid (HA) hydrogel with spatially controlled cell-adhesion properties based on photo-polymerisation cross-linking and functionalization. The approach utilises the same reaction pathway for both steps meaning that it is user-friendly and allows for adaptation at any stage during the fabrication process. Moreover, the process does not require any additional cross-linkers. The hydrogel is formed by UV-initiated radical addition reaction between acrylamide (Am) groups on the HA backbone. Cell adhesion is modulated by functionalising the adhesion peptide sequence arginine-glycine-aspartate onto the hydrogel surface via radical mediated thiol-ene reaction using the non-reacted Am groups. We show that 10 × 10 µm2 squares could be patterned with sharp features and a good resolution. The smallest area that could be patterned resulting in good cell adhesion was 25 × 25 µm2 squares, showing single-cell adhesion. Mouse brain endothelial cells adhered and remained in culture for up to 7 days on 100 × 100 µm2 square patterns. We see potential for this material combination for future use in novel organ-on-chip models and tissue engineering where the location of the cells is of importance and to further study endothelial cell biology.