Cotton wastes from cotton yarn spinning mills has been utilized as an alternative resource for the production of Microcrystalline Cellulose (MCC), an important ingredient in food, cosmetic, pharmaceutical industries, etc. The main processes conducted including cotton wastes sorting, cotton lint pretreatment, and alpha cellulose hydrolysis. In this study, several parameters on pretreatment and hydrolysis were varied in order to produce MCC which characteristics were evaluated comparing to Avicel PH-101, a commercial MCC product. Pretreatment was carried out using 17.5% alkali (w/v) at temperature 80 o C for 30 minutes, followed by a bleaching process at 5-30% H2O2 (v/v). The hydrolysis reaction was done using 1.25 M sulfuric acid, at temperature 80 o C and 96 o C for 2-4 hours. Results showed that pretreatment process with alkali for 30 minutes followed by a bleaching process with 5% H2O2 (v/v) was able to isolate 87% alpha cellulose with 99.98% of purity. The yields of MCC powders produced from acid hydrolysis of alpha cellulose at 96 o C for 2, 3, and 4 hours were 74.6%, 70.2%, and 42.8%, respectively. According to the SEM, infrared spectra, and XRD results, the process conducted was applicable to produce MCC that physically and chemically similar to the characteristics of Avicel PH-101.