Recently, KOH doped polybenzimidazole (PBI/KOH) membrane has been reported as polymer electrolyte membrane for alkaline direct alcohol fuel cell (ADAFC), but little is known about its durability for ADAFC application. In this paper, the durability of PBI/KOH membrane for air-breathing alkaline direct ethanol fuel cell (ADEFC) is evaluated by means of ex situ and in situ tests. In the case of ex situ durability test, the ionic conductivity of PBI/KOH degrades from initial 0.023Scm−1 to 0.01Scm−1 after 100h, and the degrading rate was 1.3×10−4 Scm−1 h−1. As for in situ test, Pt-free air-breathing ADEFC with PBI/KOH membrane can output a peak power density of 16mWcm−2 at 60°C. Moreover, it can stably operate for 336h above 0.3V. In addition, the interaction between KOH and PBI matrix is also explored by density functional theory study.