Land use change caused by human activities has a significant impact on regional ecosystem services. It is of great significance to clarify the impact mechanism of the proportion of landscape area of different land use types on ecosystem services for regional landscape regulation and land management. Here,taking the middle and lower reaches of the Yangtze River as the research area and the secondary watershed as the research unit, we evaluated the spatial distribution characteristics of water yield (WY), carbon sequestration (CS), and soil conservation (SC) of ecosystem services based on the InVEST model, the RULSE model and the NPP data.Then,piecewise linear regression was used to explore the response and threshold of total ecosystem services (TES) to the proportion of forest land, construction land and cultivated land that were strongly affected by human activities. The results displayed as follow: (1) The areas with high proportion of forest land in the middle and lower reaches of the Yangtze River are distributed in the west and south, the areas with high proportion of construction land are distributed in the east and north, and the areas with high proportion of cultivated land are distributed in the northeast and central and western regions. (2) The spatial distribution of ecosystem services showed obviously spatial heterogeneity. Among them, WY showed the distribution characteristics of high in the southwest and low in the northeast. CS showed the distribution characteristics of high in the south and low in the north. The high value area of SC is mainly distributed in mountainous areas with high vegetation coverage. The distribution of TES and SC showed similar characteristics. (3) The proportion of forest land area had a positive effect on TES, and its influence threshold was 93.2%. When the threshold is exceeded, the promotion effect will be strengthened. The proportion of construction land and cultivated land had a negative effect on TES. When the proportion of area exceeds 8.7% and 46.9% respectively, the negative effect of the two will be weakened, and the negative effect of cultivated land before and after the threshold is weaker than that of construction land. Therefore, restricting the development of construction land and cultivated land and promoting the increase of forest land area can improve the service level of watershed ecosystem. The research results can provide a theoretical basis and reference for landscape regulation and land use management in the middle and lower reaches of the Yangtze River.