Wuhan has a dense network of rivers and lakes. Due to the city’s development, the water system has been fragmented, the degradation of lakes is becoming increasingly severe, and the eco-environment has been significantly damaged. By collecting samples of the central surface water bodies in Wuhan, including Yangtze River water, Han River water, lake water, and precipitation, and by utilizing hydrogen and oxygen isotopes and multivariate statistical methods, the hydraulic connectivity and ecological environmental effects between the Yangtze River, the Han River, and the lakes were revealed. The results indicated the following: (1) The local meteoric water Line (LMWL) in the Wuhan area was δD = 7.47δ18O + 1.77. The river water line equation was approximately parallel to the atmospheric precipitation line in the Wuhan area. The intercept and slope of the lake waterline equation were significantly smaller. The enrichment degree of δ18O and δD was Yangtze River < Hanjiang River < lake water. (2) The cluster analysis showed that the lakes could be divided into two types, i.e., inner-flow degraded (IFD) lakes and outer-flow ecological (OFE) lakes. Urban expansion has resulted in fragmentation of the IFD lakes, changing the connectivity between rivers and lakes and weakening the exchange of water bodies between the Yangtze River and lakes. Simultaneously, evaporation has caused hydrogen and oxygen isotope fractionation, resulting in the relative enrichment of isotopes. The IFD lakes included the Taizi Lake, Yehu Lake, and the Shenshan Lake. The OFE lakes and the Yangtze River were active, evaporation was weak, and the hydrogen and oxygen isotopes were relatively depleted, mainly including the Huangjia Lake, the East Lake, the Tangxun Lake, etc. (3) The excessive deuterium (d-excess) parameter values in the Yangtze River and the Han River water were positive. In contrast, the d values in the lakes were mainly negative. In the case of a weakened water cycle, the effect of evaporation enrichment on lake water δ18O and δD had a significant impact. It is suggested that the water system connection project of “North Taizi Lake-South Taizi Lake-Yangtze River” and the small lakes connecting to large lakes project of “Wild Lake-Shenshan Lake-Tangxun Lake” should be implemented in time to restore the water eco-environment.