The connectivity of large rivers is impaired by human activities, which could cause severe consequences to hydrodynamics, water quality. To clarify the changes in hydrological connectivity of large rivers and their effects on nutrients, a composite index method of river connectivity in longitudinal, lateral and vertical dimensions was developed in this study. Taking the middle reaches of the Yangtze River as an example, the hydrological connectivity of typical hydrological years and periods in the region was analyzed, and the responses of critical nutrient indexes to varied river connectivity was assessed. The results showed that significant temporal and spatial variations in river connectivity were observed, with better river connectivity in the wet year than the dry year, and worse connectivity in headwaters, tributaries, or downstream of gates and dams than other river reaches. This was mainly due to the degree of river fragmentation (DOF) and the degree of regulation (DOR) with a relative importance of 81.28% and 56.07%, respectively, implying that gate and dam interception was the main cause of impaired river connectivity in the study area. Statistical methods indicated that the impaired connectivity in the middle reaches of the Yangtze River implied a stronger retention of nutrients by dams, and that retention effect was greater during the flood period than the non-flood period. The stronger negative correlation between connectivity and TP (correlation coefficient = −0.58) than TN (correlation coefficient = −0.46) during the flood period revealed that phosphorus was more influenced by river connectivity. The results of this study provide important information on water resources and water quality for the management of large rivers.