Three-dimensional material microstructuring by femtosecond laser-induced two-photon polymerization is emerging as an important tool in biomedicine. During two-photon polymerization, a tightly focused femtosecond laser pulse induces a crosslinking photoreaction in the polymer confined within the focal volume. As a rapid-prototyping technique, two-photon polymerization enables the fabrication of truly arbitrary three-dimensional micro- and nano-structures directly from computer models, with a spatial resolution down to 100 nm. In this review, we discuss the fundamentals, experimental methods, and materials used for two-photon polymerization; in addition, we present some applications of this technology related to microfluidics and to biomaterial scaffolds for tissue engineering and regenerative medicine.