Single-layer MoS2 and Ni80Cr20, as well as a double-layer Ni80Cr20/MoS2, films were fabricated on pure iron substrate by magnetron sputtering, and their hydrogen permeation properties were investigated by electrochemical hydrogen permeation experiments. It was found that the single-layer MoS2 or Ni80Cr20 could act as a barrier effectively retarding hydrogen permeation toward the substrate due to their lower diffusion coefficient. Using the overpotential stepping hydrogen permeation test (OSHPT), the lattice diffusion coefficient (DL) of hydrogen in MoS2 and Ni80Cr20 films was determined to be 8.35 × 10−8 and 1.76 × 10−9 cm2/s, respectively. Furthermore, it was observed that the use of Ni80Cr20 as the intermediate layer not only could improve the surface state of MoS2 after hydrogen permeation but also enhanced the effect of retarding hydrogen permeation in the double-layer Ni80Cr20/MoS2 film. In addition, the influences of MoS2 and Ni80Cr20 films on the hydrogen evolution reaction (HER) were also investigated.