Ecological limits of hydrological alteration (ELOHA) framework aims to set limits of hydrologic alteration based on empirical hydroecological relationships built along flow impairment gradients. These relationships can be specific for different natural flow regime types. The object of our study is testing the ELOHA method in the Ebro River Basin. Seven flow alteration–ecological response curves were obtained in the present study as preliminary guidelines for developing and calibrating environmental flow standards in the Ebro River Basin. For continental Mediterranean‐pluvial flow regime mean monthly flow in March was related to Plecoptera abundance (ELOHAs ±21% from baseline), and high flow magnitude and frequency were closely related to Plecoptera, Ephemeroptera and Trichoptera richness (ELOHAs for high flow magnitude and frequency were +18 and +54% the reference central value respectively). For nivo‐pluvial flow regime, high flow magnitude was found indicative of macroinvertebrate biodiversity and Coleoptera abundance (ELOHAS −12 and +23% from central reference value respectively). And for pluvio‐oceanic flow regime, mean monthly flow in January was indicative of the proportion of Gastropoda, Oligochaeta and Diptera taxa (GOLD index) (ELOHA +17% from baseline). These hydrological limits are endorsed by recent international reference works. Flow alteration–ecological relationships presented here were constructed from real monthly flow data records and macroinvertebrate samples at family level. ELOHA values obtained in this study pretend to be a starting point for implementing an environmental flow regime program in the Ebro River Basin. Copyright © 2016 John Wiley & Sons, Ltd.