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In vanadium redox flow batteries (VRFBs), simultaneously achieving high proton conductivity, low vanadium-ion permeability, and outstanding chemical stability using electrolyte membranes is a significant challenge. In this study, we report the fabrication of a tri-directional poly(2,5-benzimidazole) (T-ABPBI) membrane using a direct casting method. The direct-cast T-ABPBI (D-T-ABPBI) membrane was fabricated by modifying the microstructure of the membrane while retaining the chemical structure of ABPBI, having outstanding chemical stability. The D-T-ABPBI membrane exhibited lower crystallinity and an expanded free volume compared to the general solvent-cast T-ABPBI (S-T-ABPBI) membrane, resulting in enhanced hydrophilic absorption capabilities. Compared to the S-T-ABPBI membrane, the enhanced hydrophilic absorption capability of the D-T-ABPBI membrane resulted in a decrease in the specific resistance (the area-specific resistance of S-T-ABPBI and D-T-ABPBI membrane is 1.75 and 0.98 Ωcm², respectively). Additionally, the D-T-ABPBI membrane showed lower vanadium permeability (3.40 × 10⁻⁷ cm² min⁻¹) compared to that of Nafion 115 (5.20 × 10⁻⁷ cm² min⁻¹) due to the Donnan exclusion effect. Owing to the synergistic effects of these properties, the VRFB assembled with D-T-ABPBI membrane had higher or equivalent coulomb efficiencies (>97%) and energy efficiencies (70–91%) than Nafion 115 at various current densities (200–40 mA cm⁻²). Furthermore, the D-T-ABPBI membrane exhibited stable performance for over 300 cycles at 100 mA cm⁻², suggesting its outstanding chemical stability against the highly oxidizing VO₂⁺ ions during practical VRFB operation. These results indicate that the newly fabricated D-T-ABPBI membranes are promising candidates for VRFB application.