The key to enhancing the activation efficiency of peroxydisulfate (PDS) lies in facilitating the adsorption of PDS and accelerating the rate of electron transfer at the interface. In this work, the oxygen vacancy (Ovs) concentration of ultrathin Bi4Ti3O12 (BTO) was controlled in situ by high-energy electron beam bombardment. BTOV-300 exhibited a significant activation effect on PDS. The degradation rate constant (kobs) for the removal of ciprofloxacin (CIP) was measured to be 0.2080 min−1 under visible light irradiation, which was 3.71 times greater than that of the original BTO. Density functional theory calculations uncovered the principal role of oxygen vacancies as active sites for PDS activation. Introducing OVs significantly boosted the adsorption energy of PDS on BTO and lengthened the OO bond of PDS. This study represents the potential mechanism behind the activation of PDS by BTOV and provides valuable insights for enhancing the photoactivation efficiency of PDS.