A passive air-breathing liquid feed direct methanol fuel cell with a 100cm2 active area without any auxiliary device is designed and fabricated. Then the effect of methanol concentration on the performance of fabricated passive DMFC is investigated experimentally. The results show that there is an optimum value for the methanol concentration that yields the best performance. Consequently, in this study, 4M is proved to be the optimal methanol concentration. Furthermore, the effect of the location and number of gasket and also the effect of cathode end frame properties are tested experimentally. The obtained results indicate that the number and location of gaskets have significant effects on cell performance. Also, according to results, it can be concluded that a high rigidity and less flexibility of cathode end frame material leads to a better cell performance. Finally, according to experimental results, with an optimized design, a maximum power output of 520mW (5.2mWcm−2) is achieved under ambient conditions.