A polyfluorene-molybdenum oxide nanocomposite (PF-MoO3) was successfully prepared in swollen liquid crystalline (SLC) lamellar phase. The morphology, shape, and structure of the nanocomposite are characterized by field emission scanning electron microscopy (FESEM), X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The obtained PF-MoO3 material was loaded over a glassy carbon electrode (GCE). The PF-MoO3/GCE was employed as a working electrode for the detection of glucose by differential pulse voltammetry (DPV) and cyclic voltammetry (CV) techniques. The determined limits of detection (LOD) and the limits of quantification (LOQ) from CV were 7.90×10-5 M and 2.63×10-5 M, respectively. The calculated sensitivity of the PF-MoO3 electrode material for glucose was estimated to be 4.29×104 µA L mol-1 cm-2. The values of LOD and LOQ obtained from DPV data were 7.05×10-5 M and 2.35×10-5 M, respectively. The results were in agreement with CV observations. Similarly, the glucose sensitivity for the PF-MoO3/GCE by DPV technique was 5.18×103 µA L mol-1 cm-2. In this research, we have developed a highly sensitive glucose sensor by modification of the GCE electrode surface with PF-MoO3 nanocomposite.