Periodate-based advanced oxidation processes have been diffusely practiced for pollutant decontamination. However, the thermo-activation of periodate process (heat/PI), an effective water pollution removal process, has been rarely discussed, and the degradation pathway of this heat/PI system requires investigation. In this work, tetracycline antibiotics were selected as the model micropollutant for the comprehensive evaluation of the heat/PI system. The heat/PI system exhibited good performance for tetracycline (TC) remediation with temperature increases. The principal reactive oxidative species in the heat/PI system was confirmed using quenching experiments and electron paramagnetic resonance experiments. Further, the potential reactive sites in the TC were identified based on the density functional theory calculation. Based on the detection results of intermediates, there was no significant difference in byproducts generated during TC degradation under various temperatures in the heat/PI system. The Toxicity Estimation Software Tool (T.E.S.T.) method was applied to calculate the individual toxicity of the byproducts. This study contributes to a comprehensive explanation of the process of the thermal activation of periodate, and in particular, it explains the source of oxidation power, the transformation of byproducts, and the toxicity of reaction systems.