Objective Hemoglobin is the iron-containing protein in the red blood cells of many animals. The primary function of hemoglobin is to transport oxygen from lung to tissues. It is composed of two identical α-globin subunits and two identical β-globin subunits. Hemoglobin has unique magnetic properties. The paramagnetism of deoxyhemoglobin, and the diamagnetism of oxyhemoglobin and carboxyhaemoglobin have been reported previously. Studies have also shown that external magnetic field affected blood flow rate, but whether magnetic field may affect the oxygenation rate of hemoglobin remains unknown. Here in this study, we are aiming to address this question with recombinant hemoglobin. Human hemoglobin and yak hemoglobin were selected as the research objects, and a recombinant protein expression and purification system was established to explore the magnetic field effects on the oxygenation rate of hemoglobin, as well as the differences in the oxygenation rate between human hemoglobin and yak hemoglobin under external magnetic field. Methods The recombinant expression and purification system of human and yak hemoglobin was established. The recombinant hemoglobin expression was further optimized and appropriated inducing temperature and IPTG concentration were screened. Recombinant human hemoglobin and yak hemoglobin were purified to homogeneity by affinity chromatography and further by size-exclusion chromatography. SDS-PAGE was used to validate the purification, and UV-Vis spectrum and EPR were used to characterize the biochemical properties of recombinant hemoglobin. Deoxyhemoglobin of human and yak were placed under 0.3 T external magnetic field to test the magnetic field effects on oxygenation rate, and geomagnetic field condition was used as a sham control. The UV-vis spectrum data were measured every 10 minutes, and the concentration and proportion of oxygenated hemoglobin, deoxyhemoglobin and methemoglobin were calculated to analyze the effects of magnetic field on the oxygenation rate of hemoglobin. The magnetic properties of human oxygenated hemoglobin and human deoxygenated hemoglobin have been measured by SQUID, a superconducting quantum interference magnetic measurement system. Three biological replications were performed for each experiment. The possible mechanism of the effect of magnetic field on the oxygenation rate of hemoglobin has been investigated and discussed. Results Human and yak hemoglobin were successfully expressed and purified by E.coli prokaryotic expression system. The optimal expression temperature was 30℃, and the most suitable IPTG concentration was 1mM. EPR results suggested that trace amount of methemoglobin existed both in the purified human hemoglobin and yak hemoglobin proteins. The oxygenation rate of yak hemoglobin appeared to be faster than that of human hemoglobin, and the additional magnetic field treatment significantly increased the oxygenation rate of both human and yak hemoglobin, and yak hemoglobin was more sensitive to magnetic field than human hemoglobin. The paramagnetism of deoxyhemoglobin was verified by SQUID measurement. However, the diamagnetism of oxygenated hemoglobin remains uncertain, probably due to the presence of trace amount of methemoglobin in the sample of oxygenated hemoglobin, which was consistent with EPR results. Conclusion In this study, human and yak hemoglobin were successfully expressed and purified. The purified hemoglobin proteins have similar function and conformational states as native protein. External static magnetic fields facilitate hemoglobin oxygenation, and yak hemoglobin seems more sensitive to magnetic field compared with human hemoglobin. These findings provide theoretical basis for the potential applications of applying magnetic field to improve hypoxia symptoms in clinical practice in the future.