Nanozyme-based colorimetric sensors have received considerable attention due to their unique properties. The size, shape, and surface chemistry of these nanozymes could dramatically influence their sensing behaviors. Herein, a comparative study of VO2 nanoparticles with different morphologies (nanofibers, nanosheets, and nanorods) was conducted and applied to the sensitive colorimetric detection of H2O2 and glucose. The peroxidase-like activities and mechanisms of VO2 nanoparticles were analyzed. Among the VO2 nanoparticles, VO2 nanofibers exhibited the best peroxidase-like activity. Finally, a comparative quantitative detections of H2O2 and glucose were done on fiber, sheet, and rod nanoparticles. Under the optimal reaction conditions, the lower limit of detection (LOD) of the VO2 nanofibers, nanosheets, and nanorods for H2O2 are found to be 0.018, 0.266, and 0.41 mM, respectively. The VO2 nanofibers, nanosheets, and nanorods show the linear response for H2O2 from 0.025–10, 0.488–62.5, and 0.488–15.625 mM, respectively. The lower limit of detection (LOD) of the VO2 nanofibers, nanosheets, and nanorods for glucose are found to be 0.009, 0.348, and 0.437 mM, respectively. The VO2 nanofibers, nanosheets, and nanorods show the linear response for glucose from 0.01–10, 0.625–15, and 0.625–10 mM, respectively. The proposed work will contribute to the nanozyme-based colorimetric assay.