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Bacterial biofilm causes severe antibiotic resistance. An extracellular polymeric substance (EPS) is the main component in the bacterial biofilm. Alginate is a key EPS component in the biofilm of <i>Pseudomonas aeruginosa</i> and responsible for surface adhesion and stabilization of biofilm. Alginate lyase has emerged as an efficient therapeutic strategy targeting to degrade the alginate in the biofilm of <i>P. aeruginosa</i>. However, the application of this enzyme is limited by its poor stability. In this study, chitosan nanoparticles (CS-NPs) were synthesized using low molecular weight chitosan and alginate lyase Aly08 was immobilized on low molecular weight chitosan nanoparticles (AL-LMW-CS-NPs). As a result, the immobilization significantly enhanced the thermal stability and reusability of Aly08. In addition, compared with free Aly08, the immobilized AL-LMW-CS-NPs exhibited higher efficiency in inhibiting biofilm formation and interrupting the established mature biofilm of <i>P. aeruginosa</i>, which could reduce its biomass and thickness confirmed by confocal microscopy. Moreover, the biofilm disruption greatly increased the antibiotic sensitivity of <i>P. aeruginosa</i>. This research will contribute to the further development of alginate lyase as an anti-biofilm agent.