Enterobacter aerogenes cells were mutated by nuclear irradiation of 60Co γ-rays. E. aerogenes mutants were screened with larger colour circles of acid by-products. Hydrogenase activity of the E. aerogenes ZJU1 mutant improved by 75.3%. Hydrogen yield (301 mL H2/g glucose) with mutant was higher by 81.8% than the wild.