Cellulose is one of the most abundant molecules on the planet that presence in all plant cell walls on Napier grass and bamboo. Cellulose has been extensively used in industries like textile, food, composites, and pharmaceuticals, due to its outstanding properties. This research project aims to obtain a yield of cellulose from Napier grass, bamboo and Kenaf fiber by manipulating the operating condition of alkaline treatment. The extracted cellulose fiber was then being characterized to identify its yield, chemical composition, and crystallinity of the fiber by applying moisture content, chemical analysis, FTIR and X-ray Diffraction. As the temperature of the alkali reaction increases, the cellulose yield decreases due to the formation of purified cellulose after chemical process. Cellulose content increased after the fibers were water retted. The results were then validated by using FTIR analysis, which the amount of hemicellulose and lignin content decreased as the concentration, temperature, and time increases after treatment and the peak intensity band for lignin and hemicellulose reduces which indicate the removal of these two components. XRD analysis showed a declining trend for its crystallinity index as the temperature elevates. The crystallinity of the treated fibers did not affect the crystalline structure of the cellulose and that water retted fibers have higher crystallinity index. © 2023, Association of American Publishers. All rights reserved.