Microcrystalline cellulose (MCC) is a green material that has widespread applications in pharmaceuticals, food, cosmetics, and other industries because of its biocompatibility, biodegradability, hydrophilicity, and acid-insolubility. In this study, MCC was prepared from cotton waste via alkaline treatment and sulfuric acid hydrolysis. Further, the synthesized cotton-based MCC was characterized using Fourier transform infrared (FTIR), X-ray photoelectron, and energy dispersive X-ray spectroscopies. Based on these results, the major components were identified as carbon and oxygen. This finding was evidenced by the FTIR analysis, which displayed peak wavenumbers at 3446.9, 2891.1, 1649.5, 1380.1, 1061.2, and 1050 to 1150 cm-1. The surface morphology was also examined by field emission scanning electron microscopy and field emission transmission electron microscopy, which showed that the prepared MCC has a smooth surface and a consistent, rod-like shape. In addition, the MCC exhibited the typical diffraction peaks of a crystalline structure of cellulose II at 12.2°, 20°, and 22.03°, which correspond to the diffraction planes of 1-10, 110, and 020, respectively, and had a crystallinity index of 78.7%. Moreover, the prepared MCC had a diameter of 37.8 µm and exhibited good stability with a peak at -76.5 mV. Further, the cotton-based MCC exhibited high thermal stability, as revealed by the TGA.