Organ-on-chip platforms aim to improve preclinical models for organ-level responses to novel drug compounds. Heart-on-a-chip assays in particular require tissue engineering techniques that rely on labor-intensive photolithographic fabrication or resolution-limited 3D printing of micropatterned substrates, which limits turnover and flexibility of prototyping. We present a rapid and automated method for large scale on-demand micropatterning of gelatin hydrogels for organ-on-chip applications using a novel biocompatible laser-etching approach. Fast and automated micropatterning is achieved via photosensitization of gelatin using riboflavin-5'phosphate followed by UV laser-mediated photoablation of the gel surface in user-defined patterns only limited by the resolution of the 15 μm wide laser focal point. Using this photopatterning approach, we generated microscale surface groove and pillar structures with feature dimensions on the order of 10-30 μm. The standard deviation of feature height was 0.3 μm, demonstrating robustness and reproducibility. Importantly, the UV-patterning process is non-destructive and does not alter gelatin micromechanical properties. Furthermore, as a quality control step, UV-patterned heart chip substrates were seeded with rat or human cardiac myocytes, and we verified that the resulting cardiac tissues achieved structural organization, contractile function, and long-term viability comparable to manually patterned gelatin substrates. Start-to-finish, UV-patterning shortened the time required to design and manufacture micropatterned gelatin substrates for heart-on-chip applications by up to 60% compared to traditional lithography-based approaches, providing an important technological advance enroute to automated and continuous manufacturing of organ-on-chips.