The present investigation compares the diverse methods of cellulose extraction from rice straw. Furthermore, the purified cellulosic material was utilized for the electrospinning of cellulose nanofibers. Based on the differential scanning calorimeter and Fourier transform infrared spectroscopy analyses, the new protocol was compared to the other methods showing lower amorphous structure and also lower lignin and hemicellulose in crystalline α-cellulose structure. The protocol, which included ultrasonic mechanical treatment, showed a higher crystallinity of the corresponding cellulose giving microfibers of 2.9 ± 0.2 μm in average diameter based on the scanning electron microscope images. Cellulose nanofiber was prepared from its solution in trifluoroacetic acid using general one-step electrospinning process. The simultaneous effects of four processing variables including solution concentration (C), applied voltage (V), spinning distance (d), and volume flow rate (Q) on mean fiber diameter (MFD) and standard deviation of fiber diameter (StdFD) were investigated quantitatively and qualitatively. A range of MFD between 96 ± 26 nm and 292 ± 35 nm was recorded for further analysis. The response surface methodology was employed to establish quadratic models for MFD and StdFD. 2 adj R was found to be 96.18% and 91.25%, respectively, for the MFD and StdFD models, showing the good prediction ability of the models. The response surface plots showed strong relationship among variables.