Fabrication of three-dimensional (3D) hydrogel microenvironments with predefined geometry and porosity can facilitate important requirements in tissue engineering and regenerative medicine. Chitosan (CH) is well known as a biocompatible hydrogel with prospective biological properties for biomedical aims. So far, microstructuring of this soft material presents a great limitation for its application as functional supporting material for guided tissue formation. Enabling photopolymerization, chemically modified CH can be applied for the biofabrication of reproducible 3D scaffolds using rapid prototyping techniques like two-photon polymerization (2PP) or others. The application of this technique allows precise serial fabrication of computer-designed microstructure geometries by scanning a femtosecond laser beam within a photosensitive material. This work explores a new synthesis of water-soluble photosensitive chitosan and the fabrication of well-defined microstructures from the generated materials. To modulate the mechanical and biochemical properties of the material, CH was combined and cross-linked with synthetic poly(ethylene glycol) diacrylate. For a biological adaption to the in vivo situation, CH was covalently crosslinked with a photosensitive modified vascular endothelial growth factor (VEGF). Performed in vitro studies reveal that modified CH is biocompatible. VEGF enhances CH bioactivity. Furthermore, a 3D CH scaffold can be successfully seeded with cells. Therefore, the established CH holds great promise for future applications in tissue engineering.