Famous for Taklimakan, the world’s second largest sandy desert, the Tarim Basin in Xinjiang has long attracted researchers from various fields to investigate its paleoenvironment and antiquity. The southern part of this basin is an ideal region in which to investigate the interactions between humans and the environment due to its fragile habitat and prosperous ancient civilizations. However, the lack of direct radiocarbon dating data has caused the chronologies of some of the archaeological sites to be debatable, which hinders our ability to reconstruct historical patterns of human activity and further understand, in a coherent manner, their interaction with the environment. This study reports 25 new radiocarbon dates acquired from ten undated archaeological sites in the southern Taklimakan Desert in order to refine their chronologies. Based on this, a radiocarbon dataset was established to reveal the trajectory of human activity with the support of Bayesian chronological modeling. The results indicate a two-millennium continuous flourishing of the local society since the beginning of the first millennium BCE, as well as a peak of human activity during the Tang Dynasty (618–907 CE). The distinct trajectory of human activity in the southern Tarim Basin revealed by this study provides a solid foundation for further assessments of human–environment interaction in the Tarim Basin and along the Silk Road.