Unearthing an ideal model for disclosing the role of defect sites in solar CO2 reduction remains a great challenge. Here, freestanding gram-scale single-unit-cell o-BiVO4 layers are successfully synthesized for the first time. Positron annihilation spectrometry and X-ray fluorescence unveil their distinct vanadium vacancy concentrations. Density functional calculations reveal that the introduction of vanadium vacancies brings a new defect level and higher hole concentration near Fermi level, resulting in increased photoabsorption and superior electronic conductivity. The higher surface photovoltage intensity of single-unit-cell o-BiVO4 layers with rich vanadium vacancies ensures their higher carriers separation efficiency, further confirmed by the increased carriers lifetime from 74.5 to 143.6 ns revealed by time-resolved fluorescence emission decay spectra. As a result, single-unit-cell o-BiVO4 layers with rich vanadium vacancies exhibit a high methanol formation rate up to 398.3 μmol g-1 h-1 and an apparent quantum efficiency of 5.96% at 350 nm, much larger than that of single-unit-cell o-BiVO4 layers with poor vanadium vacancies, and also the former's catalytic activity proceeds without deactivation even after 96 h. This highly efficient and spectrally stable CO2 photoconversion performances hold great promise for practical implementation of solar fuel production.