Recently, smart biomaterials have gained much attention owing to their desired responsive behavior to environmental changes such as pH, ions etc. Among the various smart biomaterials created by different methods, photo-mediated biomaterials are important for achieving 3D structures with precise configuration. In this chapter, a new fabrication method, 3D printing technology related to two-photon polymerization which can precisely create 3D photo-mediated biomaterial scaffolds, will be introduced. The principle of this 3D printing technique, as well as the design strategies of photo-mediated biomaterials for tissue engineering in biology will be reviewed. With the help of photo-mediated biomaterials, living cells’ functions are able to be monitored by changes in the surrounding microenvironment. A better understanding of the recent development of photo-mediated biomaterial scaffolds for tissue engineering has the potential to promote the scientific fundamentals for biological applications.