River-floodplain systems are amongst the most productive—but often severely impacted—aquatic systems worldwide. We explored the ecological response of fish to flow regime in a large river-floodplain system by studying the relationships between (1) discharge and inundated floodplain area, with a focus on spatial and temporal patterns in floodplain lake connectivity, and (2) flood volume and fisheries catch. Our results demonstrate a non-linear relationship between discharge and floodplain inundation with considerable hysteresis due to differences in inundation and drainage rate. Inundation extent was mostly determined by flood volume, not peak discharge. We found that the more isolated lakes (that is, lakes with a shorter connection duration to the river) are located at higher local elevation and at larger hydrological distance from the main rivers: geographical distance to the river appears a poor predictor of lake isolation. Although year-to-year fish catches in the floodplain were significantly larger with larger flood volumes in the floodplain, they were not in the main river, suggesting that mechanisms that increase catch, such as increased floodplain access or increased somatic growth, are stimulated by flooding in the floodplain, but not in the river. Fish species that profit from flooding belong to different feeding guilds, suggesting that all trophic levels may benefit from flooding. We found indications that the ecological functioning of floodplains is not limited to its temporary availability as habitat. Refugia can be present within the floodplain itself, which should be considered in the management of large rivers and their floodplain.