Abstract:[Objective] Extracellular polysaccharides, indispensable components of biofilm, play a role in bacterial pathogenicity and antibiotic resistance. Targeted elimination of the core extracellular polysaccharides of biofilm by enzyme preparations can fundamentally destroy the core skeleton of the biofilm and help overcome the harm caused by biofilm. [Methods] On the basis of the rich gene sequence information in the NCBI database, we screened the hydrolases targeting the extracellular polysaccharides Pel, Psl, algin, Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG), and cellulose in the biofilm. Further, we employed the bioinformatics tools such as phyre2 and SWISS-MODEL to analyze the physical and chemical properties, genetic evolution, functional domains, and three-dimensional structures of these hydrolases. [Results] A total of 153 hydrolases targeting extracellular polysaccharides in biofilm were screened out and their sequence information was obtained. Among them, 30, 25, 33, 30, and 35 hydrolases targeted Pel, Psl, algin, PANG, and cellulose, respectively, which belonged to the glycoside-hydrolase family GH114, Glyco_hydro superfamily, AlgLyase superfamily, glycoside-hydrolase family GH13, and glycosyl hydrolase family GH8.[Conclusion] These hydrolases have the potential to cleavage the core extracellular polysaccharides in biofilm and need to be further developed and applied. This study provides the most comprehensive sequence and biological information of hydrolases targeting core extracellular polysaccharides in biofilms so far, laying a solid data foundation for precise prevention and targeted control of biofilms.