Gannan region, an important ecological barrier in southern China, is a typical mountain-hill area in Jiangxi Province, China, and its ecological protection is of great significance. In this study, using data of 5-year intervals from 1990 to 2015, land-use types and ecosystem service value in Gannan region were evaluated on the basis of the terrain gradient by using remote-sensing, geographic information technology, and spatial statistical methods. In addition, human interference and spatial correlation with ecosystem service value were investigated using the grid method. The results showed that (1) the proportion of forests increased as the terrain gradient increased, and the other types of land use have gradually decreased, especially forest and farmland. (2) The ecosystem service value in the Gannan region declined over the study period, and per unit area ecosystem service value reduced as the terrain gradient increased. Especially, terrain gradient 1, where the terrain index is between 0.2164 and 0.6826, was extremely decreased. (3) Spatial analysis of human interference grading showed that 90% of high human interference was distributed in the area of terrain gradient 1, where the terrain features are low altitude and low slope, and only 10% were scattered in other gradients. (4) Significant spatial aggregation distributions were observed between ecosystem service value and human interference in Gannan region, and the high-low aggregation was mainly distributed in 8 counties, namely, Dayu County, Nankang City, Ganzhou City, Xinfeng County, Ruijin City, Huichang County, Xingguo County, and Yudu County. The low-high aggregation was mainly distributed in Chongyi County and Shangyou County. The ecosystem service value and its spatial response to human interference were analyzed on the basis of the terrain gradient in Gannan region, and the results provided a theoretical basis and decision support mechanism for the rational layout and protection of ecological land in the hilly areas of southern China.