Abstract: In recent years, the advanced oxidation of peroxymonosulfate (PMS) activated by single-atom catalysts (SACs) has garnered significant attention due to its efficacy in the stable treatment of persistent organic pollutants in wastewater. However, the underlying mechanisms driving the activation of PMS by SACs to generate reactive oxidative species (ROS), as well as the structure-performance relationships, remain poorly understood. This study presents the synthesis of Ni/MoS₂ monoatomic catalysts, featuring uniformly dispersed Ni atoms, through a hydrothermal method utilizing MoS₂ as the substrate. We conducted an in-depth analysis of the intrinsic mechanisms by which this catalyst activates PMS for the degradation of tetracycline (TC). The results indicate that the Ni_0.2/MoS₂ single-atom catalyst achieved a TC degradation rate of 88.1% within 60 min, which is 3.2 times higher than that of MoS₂. Both experimental findings and density functional theory (DFT) calculations reveal that the incorporation of Ni atoms reduces the adsorption energy barrier of PMS, thereby enhancing the generation of ROS and significantly improving catalytic efficiency. Furthermore, the Ni_0.2@MoS₂ catalyst exhibits a more stable structure and superior cycling stability. This paper provides a comprehensive theoretical framework for the doping of heteroatoms in MoS₂ nanomaterials and offers a novel strategy for the development of efficient PMS activators.