Alginate lyases are commonly employed for producing alginate oligosaccharides (AOS), but their industrial application is often constrained by low thermal stability and catalytic efficiency. This study engineered mutants of alginate lyase Alyw203 from marine Vibrio based on B-factor values and negative ΔΔG values. The L172V mutant exhibited a 2.43-fold increase in half-life at 40 °C, reduced K_m (from 107 to 65 mg/mL), and enhanced k_cat/K_m (from 0.07 to 0.35 mL/mg/s), indicating improved thermal stability, substrate affinity, and catalytic efficiency. Molecular dynamics simulations revealed that these improvements originated from reconstructed hydrogen bond networks, which stabilized enzyme–substrate interactions and reduced conformational flexibility. These results demonstrate that rational design focused on strengthening hydrogen bonding can simultaneously improve both stability and activity, offering a promising strategy for industrial AOS production.