The design flood estimation should consider the regulation of upstream cascade reservoirs which has significantly altered downstream hydrologic regime. The current flood regional composition (FRC) methods do not consider the unfavorable reservoir operation conditions for flood prevention. In this paper, a novel framework, the most unfavorable flood regional composition (MUFRC) method, was proposed based on the flood risk analysis to estimate design flood in cascade reservoir operation period. The results of case study in Yalong River basin indicated that: (1) The proposed MUFRC method would allocate more flood volume into downstream uncontrolled sub-basin, and the precise definition of flood loss could make great difference on MUFRC method for rational estimation of design flood. (2) The 1000-year design flood peak, 3-day and 7-day flood volume of the outlet section estimated by MUFRC method are 15400 m3/s, 3.91, and 8.42 billion m3 respectively, which are all greater than values estimated by other FRC methods; (3) The reduction of design floods in reservoir operation period result the adjustment of flood control water level in downstream reservoirs, which could additionally generate 460 million kW·h (+1.82%) hydropower during flood season. Comparison study and sensitivity analysis further proves that the MUFRC method could rationally allocate flood volume and meanwhile balance the flood prevention risk and comprehensive utilization benefits, which is worth for further study and practical application.