Here we report a simple, rapid, and environmentally friendly method for the preparation of non-stoichiometric MoO3 (MoO3-x) nanosheets using ascorbic acid (AA) as a reducing agent under an acidic environment at room temperature. The resultant MoO3-x nanosheets show an intense blue color and strong localized surface plasmon resonance (LSPR) in the visible and near-infrared region, which can be tuned by changing the pH values of the preparation solution. The hydrogen ions intercalate into the lattice of MoO3 and bond to oxygen atoms to generate water molecules, and the electron of the AA is transferred to Mo cations, which results in the reduction of Mo6+ and formation of oxygen vacancies, giving rise to the LSPR. Additionally, a visual colorimetric assay has been developed for the detection of AA in aqueous solutions and serum samples. The absorbance at 820 nm is linearly related to the logarithm of the AA concentration in the range of 1 mM–100 mM with a limit of detection of 0.09 mM at a signal-to-noise ratio of 3.