Prestress loss of CFRP during prestressing and service stage is one of the critical technical issues in concrete structures rehabilitation. In this work, the prestress loss of reinforced concrete (RC) beams strengthened by a prestressed CFRP plate subjected to sustained loading and continuous wetting condition were experimentally studied. Eight RC beams strengthened by prestressed CFRP plate were prepared with different prestress levels of 20% and 40% respectively. The initial prestress loss was monitored up to 28 days and the loss ratios of all specimens were around 5%, which was dominated by slippage of the end anchorage system. Then, the specimens were sustainedly loaded at two levels under wet or dried environments for 170 days. The average prestress loss ratios during this period were all less than 2%, which indicates that the CFRP plate end anchorage system had good long-term performance. Moreover, the adhesive bonding was weakened by the moisture penetration, and the specimens with lower prestress levels were more susceptible to the wet condition. Nevertheless, regardless of the environmental exposure, the maximum crack widths of 40% prestressed specimens remained less than 0.2 mm even subjected to high sustained load level, which is within the limitation of the prestressed concrete structures.