The load-deflection relationship is critical for the serviceability and structural durability of reinforced concrete (RC) structures strengthened with prestressed carbon fiber-reinforced polymer (CFRP) plates. This study aims to investigate the impact of prestressing levels and wetting/drying cycles (WDCs) on the flexural stiffness of prestressed CFRP-strengthened RC beams. A modified flexural stiffness prediction model was proposed by incorporating prestressing levels, interfacial bond slip, and softening after WDCs. Subsequently, four-point bending tests were conducted on 11 strengthened beams with different prestressing levels and WDCs to estimate flexural behavior. The results suggest that WDCs can adversely affect the flexural stiffness of the strengthened beams, particularly for the post-cracking stiffness of the non-prestressed specimens. However, prestressed strengthening can mitigate the stiffness degradation caused by WDCs. Moreover, a finite element (FE) model utilizing a traction separation model can accurately predict flexural stiffness. Finally, the flexural stiffness was computed using both the existing and proposed models and the proposed model provides the closest prediction of the flexural stiffness for CFRP-strengthened beams under WDC and prestressing.