The fermentative H2-producing strain Clostridium sp. H-61 was isolated from anaerobic sludge, was used as an original strain which was induced by NTG and UV for increasing and the hydrogen production ability. One of the highest efficient H2-producing mutants was named as HCM-23 with its stable hydrogen production ability. which was measured in the batch culture experiments. With the condition of 10 g/L glucose, its cumulative hydrogen yield and hydrogen production rate was 3024 mL/L and 33.19 mmol H2/g DW·h, 69.89% and 68.14% higher than that of the original strain, respectively. The terminal liquid product compositions showed that the mutant HCM-23 fermentation was ethanol type, while the original strain H-61 fermentation was butyric acid type. Varieties of parameters of hydrogen production fermentation studied, including time, carbon source, nitrogen source, glucose concentration, glucose utilization, initial pH and incubation temperature had been studied, indicated the optimum condition of hydrogen production for the mutant HCM-23 as initial pH 6.5, temperature 36℃, and the favorite substrate was sucrose. The hydrogen production characters of the mutant and the original strain were different, such as, the growth lag phase and the utilization of inorganic nitrogen source, etc. This work shows a good application potential of NTG-UV combined mutation in the biohydrogen production. And the hydrogen production mechanism and metabolic pathway should be explored furthermore.