Rural areas play an important hinterland role in social development, and the evolution of rural spatial structure has an important impact on the function of rural ecological services. Taking Nanjing as an example, this paper conducts the research on the evolution of rural regional ecological service functions based on the scope of physical rural areas. The remote sensing quantitative measurement and CASA model are used to calculate and analyze the evolution of rural regional ecological service functions in Nanjing from 1985 to 2016. The results show that: (1) From 1985 to 2000, the value of various functions of rural ecosystem services declined in varying degrees; from 2000 to 2016, the values of all kinds of functions, except that of water conservation function, still showed a downward trend. (2) In the first stage, the decline of rural ecological service function was mainly found in the south, west to south and east to south. In the second stage, except for the increase of ecosystem services in the north and northwest directions, the decline of ecosystem services in all directions decreased in varying degrees. (3) In the stage of industrialization of villages and towns, the change of land use structure within villages had a major impact on the ecological service function. In the stage of urbanization, the impact of urban expansion on rural ecological service function was the main aspect.