Carbon dots (CDs) with intriguing fluorescent property, good biocompatibility, high stability, easy interaction with substrates, are burgeoning carbon nanoparticles with large potential in various applications. Incorporating CDs into the polymer matrix is becoming a popular strategy to endow the complex with new functions. Herein, the green-synthesized CDs was integrated into the mixture of gelatin (derived from waste fish scale) and chitosan, and a multifunctional bio-nanocomposite (defined as Gelatin/Chitosan/CDs) film was developed, which showed the excellent antibacterial, antioxidant, pH-sensitivity, UV shielding, and blue-emission properties. The effects of different concentrations of CDs on the physical, mechanical, structural, and functional activity of bio-nanocomposite film were tested. Compared with the Gelatin/Chitosan film, the Gelatin/Chitosan/CDs film with an optimum addition of 20% CDs showed the enhanced antibacterial, antioxidant as well as UV shielding activities. More importantly, it was used as an effective packaging material for fish meat preservation, reducing the loss of nutritional quality consumption, extending the shelf life of food. Besides, the bio-nanocomposite films also possessed the anti-counterfeiting and pH-responsive properties due to the strong fluorescent emission of CDs, and had the great potential in developing the intelligent packaging materials. Our work shed new light on the new application of CDs and the synthesis of bio-nanocomposite film in food industry.