Aim: This research aimed to optimize suitable hydrolytic enzymes for maximizing cottonseed milk extracts for high cottonseed milk yield, protein content and low gossypol level. Methodology: Known amount of cottonseed was soaked for 90 min at 32°C and blended (cottonseed:water@1:6). Different aliquots of the blended cottonseed slurry were treated with 1% of enzymes viz., protease, cellulase and α-amylase enzyme at pH 7.0 followed by incubation at 40 and 52°C for 2.30 hr for the extraction of cottonseed milk. The enzyme activity of extracted milk was subsequently inactivated by pasteurization (90°C, 5 min). Further analysis of physico-chemical characteristics was also carried. The control sample included milk extraction from non-enzyme treated cottonseed milk extract (30±2°C). Results: Among different treatments, cottonseed milk extraction using protease enzyme at 40°C incubation showed the highest milk yield (86.71%) with the lowest sedimentation (3.72%). Further incubation 40°C and 52°C showed the highest protein content of 2.10 and 2.27 g 100 ml-1 and gossypol reduction of 40.36 and 35.22%, respectively, in the cottonseed milk extract. Meanwhile, cellulase and α-amylase enzymes treated samples at both incubation temperatures showed poor physico-chemical characteristics as compared to control. Interpretation: Protease enzyme seems to be the most suitable for optimum or higher extraction of cottonseed milk.