Cotton stalks are good raw material for bioethanol production due to its availability throughout the world, abundance, high carbohydrate content and not involved in any food chain. Due to recalcitrant nature of cotton stalks pre-treatment, hydrolysis were not effective. In the present study pre-treatment with ammonia at room temperature for 1 week period and 121oC for 60min were attempted and it is compared with the standard 0.2M NaOH treatment. 1.5% ammonia pre-treatment at room temperature for 1 week found to remove 86% of lignin and subsequently undergone 75.92% acid hydrolysis. The acid hydrolysate obtained consists less lignin and furfurals and fermented to 5.75% ethanol with 91% fermentation efficiency with saccharomyces cerevisiae. Hence dilute ammonia pre-treatment for a week was found to be effective for bioethanol production using cotton stalks.