In this study, the suitability of a mixture containing riboflavin (vitamin B2) and triethanolamine (TEOHA) as a novel biocompatible photoinitiator for two-photon polymerization (2PP) processing was investigated. Polyethylene glycol diacrylate was crosslinked using Irgacure(®) 369, Irgacure 2959 or a riboflavin-TEOHA mixture; biocompatibility of the photopolymer extract solutions was subsequently assessed via endothelial cell proliferation assay, endothelial cell viability assay and single-cell gel electrophoresis (comet) assay. Use of a riboflavin-TEOHA mixture as a photoinitiator for 2PP processing of a tissue engineering scaffold and subsequent seeding of this scaffold with GM-7373 bovine aortic endothelial cells was also demonstrated. The riboflavin-TEOHA mixture was found to produce much more biocompatible scaffolds than those produced with Irgacure 369 or Irgacure 2959. The results suggest that riboflavin is a promising component of photoinitiators for 2PP fabrication of tissue engineering scaffolds and other medically relevant structures (e.g., biomicroelectromechanical systems).