This study was conducted to increase the amount of bio-hydrogen production from microalgae(Chlorella vulgaris) in batch reactors by thermal pre-treatment. The optimization of thermal pre-treatment was conducted using statistic experimental design of response surface methodology. Two experimental parameters of temperature and reaction time were considered. The optimization condition was founded at the coded variables of <0.52, -0.07> corresponding to the experimental of heating temperature of $95.6^{circ}C$ and reaction time of 57.9 min, respectively. Under the optimal condition, the maximum hydrogen production was predicted to 25.3mL $H_2/g$ dry cell weight (dcw), which was 9.1 times higher value of control(2.8mL $H_2/g$ dcw).