This study was conducted to evaluate the influence of cellulose whiskers (CW) from cotton linter and sonication on physical properties of glycerol-plasticized tilapia gelatin films produced by casting technique. The tensile strength and elastic modulus of the films have been improved by the addition of CW, while elongation tended to be impaired by CW loading. The barrier to water vapor has also been improved by the whiskers, and the film transparency has been unaffected. The sonication improved modulus, elongation, and transparency of the films, probably by enhancing CW dispersion on the nanocomposite films, and maybe by inducing some conformational changes on the gelatin matrix. FTIR spectroscopy study on films revealed some gelatin conformational changes induced by cellulose whiskers.