Ulva lactuca is one of green macro-algae that has a significant cellulose content. This study aims to determine the effect of variations in substrate-enzyme ratio and hydrolysis time on the enzymatic hydrolysis process of cellulose extracted from Ulva lactuca to produce fermentable sugar or reducing sugar as a raw material for making bioethanol. Firstly, Liquid Hot Water (LHW) pre-treatment process was performed at the temperature of 135°C for 20 minutes; the purpose of this pre-treatment was to reduce the content of hemicellulose and to increase the cellulose content. Secondly, enzymatic hydrolysis process using cellulase enzyme was carried out, in this process citrate buffer was needed in order to stabilize the pH level during hydrolysis process. The process variables were ratio of substrate-enzyme (1:0.05; 1:0.1; 1:1.5; 1:2 and 1:2.5 w/w) and hydrolysis time (24, 48 and 72 hours) under temperature of 45°C and pH level of 5.5. The results shows that the highest reducing sugar yield is 79.7% obtained at a ratio of substrate-enzyme of 1:2.5 (w/w) for 48 hours of hydrolysis time, with the result of reducing sugar concentration is 16.2043 mg/mL.