Melanin, a kind of well-known multifunctional biomacromolecules that are widely distributed in natural sources. In this work, polyurethane (PU)/melanin nanocomposites with enhanced tensile strength and toughness were successfully fabricated via in situ polymerization. It was found that the tensile strength (σ), elongation-at-break (εmax), and toughness (W) were improved from 5.6 MPa, 770%, and 33 MJ/m3 for PU to 51.5 MPa, 1880%, and 413 MJ/m3 for PU/melanin (2 wt %) nanocomposite, respectively. Micromorphology indicated that individualized melanin nanoparticles were specifically linked to the hard domains of PU chains and fine dispersed in matrix. FTIR, DSC, and AFM results suggested melanin induced an improvement in degree of phase separation, which resulted in remarkable enhancements in mechanical properties of PU. However, with further increasing content of melanin, a relatively large-scale phase separation was formed and led to a decrease in mechanical properties of PU. In addition, interactions between melanin and hard segments of PU were increased, leading to a higher TgHS. Moreover, the dynamic mechanical properties and rheological behavior of PU/melanin nanocomposites were further investigated.