Fe(III) is widely present in non-stoichiometric pyrrhotite (Fe_1−xS, 0 < x < 0.125); however, its role in determining the catalytic activity of Fe_1−xS remains unclear. Herein, Fe_1−xS with varying Fe(III) were prepared via the sulfidation of goethite and the pyrolysis of pyrite at different temperatures, respectively, and subsequently employed to activate peroxydisulfate (PDS) for tetracycline (TC) removal. A combination of characterization and chemical analysis revealed that the Fe(III) content in sulfurized Fe_1−xS and pyrolytic Fe_1−xS decreased linearly with increasing sulfidation temperature (R² = 0.9875) and calcination temperature (R² = 0.9665), respectively. Electrochemical and zeta potential measurements showed that both the electron-transfer capability and surface electronegativity of Fe_1−xS increased with increasing Fe(III) content. Importantly, in the Fe_1−xS/PDS system, the Fe(III) content in sulfurized Fe_1−xS and pyrolytic Fe_1−xS was positively correlated with the second-order reaction rate constant (k₂, R² = 0.9276) and first-order reaction rate constant (k₁, R² = 0.9741) for TC removal, respectively, suggesting a clear structure-activity relationship between Fe(III) content and catalytic activity. Meanwhile, Fe release, reactive oxygen species (ROSs) generation, and PDS consumption increased with increasing Fe(III) content in Fe_1−xS. In addition, sulfurized Fe_1−xS and pyrolytic Fe_1−xS show a wide pH operating range and moderate reusability. These findings provide valuable insights into the rational design of Fe_1−xS-based catalysts for advanced oxidation processes to remove refractory organic pollutants.