In tissue engineering, three-dimensional scaffolds, which should ensure necessary mechanical and biological microenvironment and nutrient, oxygen and grow factor delivery to proliferating cells, are an essential element. They can be formed from polymeric, ceramic and hybrid materials via different techniques. Modern laser fabrication methods, which provide high accuracy of positioning and energy focusing and allow the precise porous scaffold formation, are particularly interesting. Two-photon polymerization is one of the most promising laser-based techniques and permits the use of a large material variety for scaffold fabrication with the possibility of controlling accurately their microarchitecture. While the number of studies on two-photon polymerization is constantly growing, it is crucial to provide a framework of its application in tissue engineering. Therefore, this chapter aims to describe recent achievements and examples of two-photon polymerization application in tissue engineering and to reveal the main trends in this field.