Air-cooled open-cathode LTPEMFC (AO-LTPEMFC) has been developed as a new power source for the portable power supply. The effect of the gas diffusion layer (GDL) on AO-LTPEMFC operation in challenging ambient air conditions was investigated in the present study. The effect of the content of polytetrafluoroethylene (PTFE) on the substrate layer and microporous layer (MPL), the thickness of GDL and the pulse width modulation (PWM) of the fan on cell performance as well as the cathode outlet surface temperature distribution were investigated. The polarization curves, electrochemical impedance spectroscopy (EIS) and dynamic voltage test were used to correlate the structural characteristics of GDL with heat dissipation, oxygen transport and water management in AO-LTPEMFC. The results showed that GDL with appropriate PTFE content in the substrate layer and MPL could significantly optimize cell performance. In addition, the thickness of GDL also had a certain impact on the mass and heat transfer of the fuel cell, while the water management in GDL was affected by PWM of the fan, simultaneously. Combined with the practice process, the optimum values of the substrate layer PTFE content, MPL layer PTFE content and thickness of GDL were identified to be 10%, 40%, and 200 μm, respectively.