Hyaluronic acid (HA) is a key glycosaminoglycan in the extracellular matrix, essential for cellular signaling, hydration, and tissue homeostasis. This study identified two novel polysaccharide lyase family 33 (PL33) hyaluronate lyases, BxHly33 and BiHly33, from the human gut microbiome using metagenomic screening. These enzymes demonstrated high specificity and stability in degrading HA, with optimal activity at pH 6.6-7.6 and temperatures of 35-40 °C. Furthermore, structural and biochemical analyses revealed their catalytic mechanisms, highlighting key residues responsible for their function. Notably, specific alanine substitutions significantly enhanced their enzymatic activity. BxHly33 and BiHly33 present promising alternatives to conventional hyaluronidases, which are often costly and immunogenic, for drug delivery and tissue engineering applications. This study will provide novel insights into exploring their therapeutic potential in HA degradation therapies.