Enfuvirtide, the inaugural biomimetic fusion inhibitor of HIV-1, has exhibited remarkable antiviral efficacy when administered in conjunction with an optimized antiretroviral regimen. Nonetheless, the high incidence (98%) of injection site reactions associated with twice-daily subcutaneous administration severely compromises patient adherence and long-term therapeutic outcomes. This study proposes hydrogel-forming microneedles (MNs) as a minimally invasive and painless modality for the transdermal delivery of this therapeutic peptide. Leveraging a rigorous Quality by Design (QbD) framework, this investigation systematically delineated the critical material attributes (CMAs) and critical process parameters (CPPs) of the hydrogel formulation, mapping their influence on the critical quality attributes (CQAs) of MNs to achieve a meticulously defined quality-target product profile (QTPP). The optimized MN formulation, achieving a desirability index of 0.871, was validated through comprehensive design space and feasibility analyses, demonstrating superior predictive accuracy and mechanical integrity. Ex vivo permeation studies elucidated the sustained and controlled release kinetics of enfuvirtide via MNs fabricated from the optimized formulation, attaining a maximum permeation of 36.26% using 11 × 11 molds, compared to 28.45% permeation observed with the control system over 24 h. Furthermore, the system's favorable swelling kinetics and enhanced viscoelastic properties significantly augmented its delivery performance relative to conventional approaches. This study not only establishes hydrogel-forming MNs as an innovative and efficacious delivery platform for enfuvirtide but also presents a robust, systematic methodology for MN development, offering transformative potential for broader pharmaceutical applications and therapeutic paradigms.