The fermentation conditions of chicken blood hemoglobin (Hb) for the preparation of small peptides by Aspergillus oryzae were optimized and the molecular weight distribution of the polypeptides were determined. On the basis of single factor experiments, response surface analysis methodology (RSM) were used to optimize the preparation conditions about Hb polypeptide. A regression model for Hb polypeptide yield was established to optimize the fermentation process and the distribution of Hb polypeptide molecular weights were determined by the gel chromatography method. The results showed that factors affected the Hb polypeptide yield were inoculation amount, culture temperature, initial pH, and the fluid volume. The optimal conditions were inoculation amount 7.9%, culture temperature 30.2℃, initial pH 7.01, and medium volume 61mL. Under these conditions, Hb polypeptide yield increased from 1.85mg/mL to 2.18mg/mL. According to the gel chromatography method, the Hb peptide molecular weights were mainly concentrated in the range of 1000-6500u, of which 1600-2500u took up the most abundant (38.39%).