Direct glycerol fuel cell with polytetrafluoroethylene thin film separator was developed. A membraneless fuel cell with a peak power density of 214.7 mW cm−2 was achieved. An 80 h durability test showed a smaller degradation rate on PTFE thin film fuel cell. The average particle size of PdAg/CNT increased from 2.9 nm to 3.7 nm during durability test.