The alpine tree line ecotone, reflecting interactions between climate and ecology, is very sensitive to climate change. To identify tree line responses to climate change, including intensity and local variations in tree line advancement, the use of Landsat images with long-term data series and fine spatial resolution is an option. However, it is a challenge to extract tree line data from Landsat images due to classification issues with outliers and temporal inconsistency. More importantly, direct classification results in sharp boundaries between forest and non-forest pixels/segments instead of representing the tree line ecotone (three ecological regions—tree species line, tree line, and timber line—are closely related to the tree line ecotone and are all significant for ecological processes). Therefore, it is important to develop a method that is able to accurately extract the tree line from Landsat images with a high temporal consistency and to identify the appropriate ecological boundary. In this study, a new methodology was developed based on the concept of a local indicator of spatial autocorrelation (LISA) to extract the tree line automatically from Landsat images. Tree line responses to climate change from 1987 to 2018 in Wuyishan National Park, China, were evaluated, and topographic effects on local variations in tree line advancement were explored. The findings supported the methodology based on the LISA concept as a valuable classifier for assessing the local spatial clusters of alpine meadows from images acquired in nongrowing seasons. The results showed that the automatically extracted line from Landsat images was the timber line due to the restriction in spatial autocorrelation. The results also indicate that parts of the tree line in the study area shifted upward vertically by 50 m under a 1 °C temperature increase during the period from 1987 to 2018, with local variations influenced by slope, elevation, and interactions with aspect. Our study contributes a novel result regarding the response of the alpine tree line to global warming in a subtropical region. Our method for automatic tree line extraction can provide fundamental information for ecosystem managers.