Constructing biopolymer-based packaging films with fantastic water resistance and mechanical properties for food preservation is highly desirable and challenging. In this work, Gliadin/Casein nanoparticles (GCNPs) were prepared by pH-driven method and embedded into konjac glucomannan/carboxymethyl chitosan (KC) film matrix to improve the water resistance and mechanical properties of KC film. Gliadin and Casein showed good compatibility and co-assembled to form compact GCNPs clusters through hydrogen bonding and hydrophobic interaction verified by FT-IR spectroscopy, and fluorescence spectroscopy. The particle size and zeta potential of GCNPs was 269.7 nm and −7.6 mV, respectively. The effect of GCNPs on the mechanics, water barrier, thermal stability, and UV-shielding of KC-GCNPs film was investigated. SEM images revealed that GCNPs uniformly distributed into KC film matrix and significantly improved the mechanics (tensile strength: 75.6 MPa, elongation at breaking: 36.7 %), water barrier ability (water contact angle: 91.3°, water vapor permeability: 0.994 g mm/m2 day kPa, water solubility: 52.0 %), thermal stability and UV blocking property of KC-GCNPs film. Furthermore, KC-GCNPs film could also be applied to extend the shelf life of grapes. This paper demonstrated the great potential of GCNPs as functional nanofillers in enhancing the physicochemical properties of KC film.