The Mo surface alloying layer was prepared on Ti6Al4V substrate using the plasma surface alloying technique. The component, structure and hardness of the Mo surface alloying layer were investigated. The impact test was used to determine the fatigue behavior of the Mo surface modified Ti6Al4V. The results showed that the Mo surface alloying layer enhanced the surface strength of Ti6Al4V substrate. The Mo surface alloying layer has a duplex structure with diffusing and coating layers. The diffusing layer in which the component and hardness change gradually could enhance load-bearing capacity to the surface coating and ensure the durability of the coating. In the impact test, the Mo modified Ti6Al4V showed excellent fatigue behavior and the cohesive failure mode was observed.