The otolith microstructure was used to identify the daily age of grass carp and silver carp larvae collected in the Jianli section of Yangtze River during 2018 to 2020, and then their hatching and spawning dates were speculated. The spawning ground and the impact of the ecological operation of the Three Gorges Reservoir on the larvae in Jianli section were also analyzed. The results showed that both the otolith microstructure and daily age composition of grass carp and silver carp larvae were similar. The age of larvae in 2018 and 2019 ranged from 8 to 15 days, about 90% of which aged 9-12 days. The day-age in 2020 ranged from 10 to 22 days, and 12-15 days of larvae were dominant. The day-age and size ranges were narrow, implying that these larvae spawned in the same or neighbor’s spawning grounds. Based on day-age and hydrologic characteristics of Yangtze River, we inferred that the Yichang section was the main spawning ground of flood larvae in Jianli section. From 2018 to 2020, the Three Gorges Reservoir carried out four ecological operations during fish spawning season, only one of which in mid-June 2018 had a corresponding larvae flood in the Jianli section. The deduced larvae flood in Jianli section based on the rest three ecological operation carried out in late May did not be found. The lower temperature and poor food in the middle reaches of the Yangtze River in late May affected the early growth and mortality of the fish larvae. Accordingly, it is suggested to strengthen the ecological operation in late June, which may play a better role in supplementing the downstream larval fish resources.