Biological hydrogen fermentation from organic substrates has been extensively studied recently. In this study, the major carbohydrate substrate from pure corn starch to kitchen waste were fed to a 10 L mesophilic continuous stirred tank reactor and a 3 L thermophilic intermittent continous stirred tank reactor to promote the process performance of anaerobic hydrogen fermentation and amylase activity. The volumetric organic loadings of mesophilic CSTR was increased by shortening hydraulic retention times from 24, 12, 9, 6 to 3 hours with feeding corn starch (12,000 mg/L) and peptone (8,000 mg/L). A series of mesophilic starch degrading fermentor operations indicated the HRT shorter than 9 hours achieving the best hydrogen fermentation, while the hydrogen production was observed and cell bound amylase was predominant. At HRT of 3 hours, the CSTR has a hydrogen production rate of 435 mmol/L/d, while the total amylase activity was about 0.5 CU/g VSS. Later, the thermophilic I-CSTR was fed with kitchen waste of two different feeding frequency daily, at the same hydrolytic retention time of 4 days. To investigate the starch hydrolysis mechanism, a time series of organic composition profile was monitored and two batch tests of white rice fermentation were also studied. According to the time series profile analysis, most of amylase was cell free type presented in fermentor of I-CSTR. With white rice fermentation, the cell-bound amylase was activated in two batch test. So, carbohydrate hydrolysis was promoted by amylase to enhance the hydrogen production.