Melanin nanoparticles (MNP) were isolated from squid ink and used for the preparation of gelatin-based nanocomposite films containing various concentration of MNP (0, 0.25, 0.5, and 1.0 wt%). The MNP was a spherical form with an average diameter of about 100 nm. The MNP was compatible with the gelatin matrix to form uniform nanocomposite films. The surface color of gelatin/MNP nanocomposite films was brown with decreased transparency, but other film properties such as mechanical, water vapor barrier, and thermal stability properties increased significantly compared with the neat gelatin film. All the film properties of the gelatin/MNP nanocomposite films were dependent on the MNP concentration. Also, the gelatin/MNP nanocomposite films exhibited a high antioxidant activity which has great potential for food packaging and biomedical applications.