【Objective】 To identify the key factors that regulate the physical vacuum stirring deoxygenation process of bovine hemoglobin and evaluate the stabilities of polyHb solution with different oxygenation degree. 【Methods】 The effects of several factors including gas liquid specific phase boundary area, pH values and polyHb concentrations on deoxygenation process were investigated through monitoring the oxygen saturation of polyHb solution. Furthermore, the MetHb contents of the studied polyHb solutions were measured at regular intervals during the experiments to evaluate the molecular stability of the polyHb through monitoring the oxidation degrees of the experimental Hb solutions under the mimic pasteurevirus inactivation conditions. 【Results】 The results showed that the more gas liquid specific phase boundary area could accelerate deoxygenation rate, the better deoxygenation results could be yielded. On the other hand, lower pH value could promote the deoxygenation process of the polyHb solution due to the decreasing of oxygen affinity of the polyhemoglobin molecules. Furthermore, results of investigation about the Hb concentration presented that Hb concentration had no remarkable effects on the deoxygenation of polyHb. In addition, the results from the stability experiments presented that higher degree of deoxygenation could decrease the oxidation process and thus increase the heat tolerance of the polyhemoglobin. 【Conclusion】 The deoxygenation of polyhemglonbin could improve the stability of the polyhemoglobin and the deoxygenation process could be regulated by changing the gas liquid specific phase boundary area and oxygen affinity. This study could provide reference and preliminary basis for the industrialization and development of HBOCs in the future.