Catalytic combustion is an important means to reduce toluene pollution, and improving the performance of catalytic combustion catalysts is of great significance for practical applications. The study of oxygen vacancies is one of the key steps to improve catalyst performance. Here, two different oxygen vacancy structures were well-defined and controllably synthesized by flame spray pyrolysis (FSP) to evaluate their effect on the catalytic combustion performance of toluene. The closely contacted oxygen vacancies (c-Vo) enhance the oxygen activation capacity of the catalyst, and the temperature of the first oxygen desorption peak and hydrogen reduction peak is 56 and 37 °C lower than the separated oxygen vacancy (s-Vo) sample, respectively. The oxygen activation energy barrier on the c-Vo is calculated to be negligible of only 0.04 eV. Both in situ DRIFT and DFT calculations indicate that the c-Vo structure accelerates the catalytic oxidation of p-toluene molecules. Moreover, due to the unique characteristics of high-temperature synthesis and rapid quenching, FSP brings excellent water resistance and high-temperature stability to the catalyst. In conclusion, utilizing the FSP in situ reduction strategy can create more c-Vo to improve the catalytic combustion performance of toluene.