Hydrogel scaffolds that mimic the native extracellular matrix (ECM) environment is a crucial part of tissue engineering. It has been demonstrated that cell behaviors can be affected by not only the hydrogel's physical and chemical properties, but also its three dimensional (3D) geometrical structures. In order to study the influence of 3D geometrical cues on cell behaviors as well as the maturation and function of engineered tissues, it is imperative to develop 3D fabrication techniques to create micro and nanoscale hydrogel constructs. Among existing techniques that can effectively pattern hydrogels, two-photon polymerization (2PP)-based femtosecond laser 3D printing technology allows one to produce hydrogel structures with 100 nm resolution. This article reviews the basics of this technique as well as some of its applications in tissue engineering.