The polyelectrolyte complexation between chitosan (CH) and chondroitin sulfate (CS) and the ultrasonication assistance were adopted as the encapsulation strategy for preparing proanthocyanidin (PC) encapsulated nanoparticles (NPs) to protect bio-functions of PC. Conditions of 1.5 mg/mL of CH and CS, 1:3 wt ratio of CH/CS, 0.5 mg/mL of PC, pH 3.25, and 1 min ultrasonic treatment were recommended by the systematic research, respectively, with NPs appearing the smallest size (∼300 nm) and high entrapment efficiency (37.5%). Ultrasound crushed aggregates into nanoparticles by the acoustic cavitation and generated more amide bonds observed by XPS. Due to the physical barrier of NPs, PC in NPs still had a 94.4% DPPH radical scavenging rate after illumination of 10 d. Application of NPs for fabricating CH bio-films was carried out, and the bio-films containing CH/CS-PC NPs decreased aerobic bacteria count and lipid oxidation of fillets stored for 8 d by 2.2 log 10 CFU/g and 51.1%, respectively, which was better than pure CH bio-films. The incorporation of PC-encapsulated NPs to CH bio-films inhibited the bacteria and oxidation induced spoilage for refrigerated grass carp flesh, providing a potential application for food preservation. • Chitosan and chondroitin sulfate formed nanoparticles to load proanthocyanidin. • Ultrasound reduced the size of particles and created more amide bonds. • The encapsulation for proanthocyanidin maintained the antioxidant effect. • Nanoparticle-contained films enhanced the quality of fish fillets during storage.