We report the growth of monoclinic MoO₃ nanorods by a simple and highly reproducible hydrothermal method. Structural and morphological studies provide significant insights about the phase and crystalline structure of the synthesized samples. Further, the non-enzymatic glucose sensing properties were investigated and the MoO₃ nanorods exhibited a sensitivity of 15.4 μA μM⁻¹ cm⁻² in the 5–175 μM linear range. Also, a quick response time of 8 s towards glucose molecules was observed, exhibiting an excellent electrochemical activity. We have also performed density functional theory (DFT) simulations to qualitatively support our experimental observations by investigating the interactions and charge-transfer mechanism of glucose on MoO₃. There is a strong interaction between glucose and the MoO₃ surface due to charge transfer from a bonded O atom of glucose to a Mo atom of MoO₃ resulting in a strong hybridization between the p orbital of O and d orbital of Mo. Thus, the MoO₃ nanorod-based electrodes are found to be good glucose sensing materials for practical industrial applications.