The effective gas diffusivity, porous structure, and tortuosity factor of catalyst layers used in proton exchange membrane fuel cells were evaluated using a microfluidic device. Sufficient gas transport properties of the catalyst layers are a key factor for achieving high-performance catalyst layers and fuel cells. In the present study, catalyst layers with different thicknesses and different carbon supports were evaluated. Stand-alone carbon black and multi-walled carbon nanotubes were blended into the catalyst layers as the support. The all-carbon-black-based catalyst layer contained some volume of isolated pores and some amount of microcracks, which depended on its thickness. The tortuosity factor was evaluated considering the effects of the isolated pores and microcracks. However, the tortuosity factor of the all-carbon-black-based catalyst layer was larger than the well-known Bruggeman-correlated tortuosity factor. When carbon nanotubes were blended into the catalyst layer, the tortuosity factor was drastically decreased to less than one-half that of the carbon-black-based catalyst layers. A change in the number of straight pores formed by the fibrous support and variation of the ionomer distribution can affect the tortuosity factor.