The influenza virus has caused a global pandemic with significant morbidity and mortality, underscoring the need to optimize antibodies for improved antiviral efficacy. The monoclonal antibody 3E1 effectively neutralizes Group 1 influenza subtypes, H1 and H5, by inhibiting acid-induced conformational changes of hemagglutinin (HA). However, its neutralizing activity is relatively weak against the Group 2 subtype, H3. In order to broaden the neutralizing activity of wild-type 3E1 (3E1-WT) against both Group 1 and Group 2 viral strains, single-point mutants (3E1-L [W32I], 3E1-H [F103I]) and a double mutant (3E1-H+L [F103I, W32I]) were designed and generated in this study. The binding affinity, microneutralizing activity, and antiviral mechanisms of the mutants were evaluated in vitro. Notably, the 3E1-L mutant exhibited significantly enhanced antiviral activity against H1N1 and H3N2 compared to 3E1-WT, inhibiting both viral entry and release. In vivo studies also indicate that 3E1-L significantly enhances both prophylactic and therapeutic efficacy against A/New-York/61/2015-CDC-LV16A (H1N1) and A/Hong Kong/17/2014/8296 (H3N2). Molecular dynamics simulations of the 3E1-L/HA complex revealed that the W32I mutation could reduce steric hindrance between tryptophan at position 32 and the complementarity-determining region L1 loop of HA. In conclusion, the W32I substitution enhances the antiviral activity of 3E1-WT, suggesting that the optimization of 3E1-L a promising strategy for the development of more effective influenza therapy and prevention.