When subjected to fatigue loading, the main failure mode of partially prestressed concrete (PPC) structure is the fatigue fracture of tensile reinforcement. Therefore, monitoring and evaluation of the steel stresses/strains in the structure are essential issues for structural design and healthy assessment. The current study experimentally investigates the possibility of using fiber Bragg grating (FBG) sensors to measure the steel strains in PPC beams in the process of fatigue loading. Six full-scale post-tensioned PPC beams were exposed to fatigue loading. Within the beams, the FBG and resistance strain gauge (RSG) sensors were independently bonded onto the surface of tensile reinforcements. A good agreement was found between the recorded results from the two different sensors. Moreover, FBG sensors show relatively good resistance to fatigue loading compared with RSG sensors, indicating that FBG sensors possess the capability for long-term health monitoring of the tensile reinforcement in PPC structures. Apart from the above findings, it can also be found that during the fatigue loading, there is stress redistribution between prestressed and non-prestressed reinforcements, and the residual strain emerges in the non-prestressed reinforcement. This phenomenon can bring about an increase of the steel stress in the non-prestressed reinforcement.