Bovine Hb-loaded nanoparticles with modulated size of pores, for use as blood substitutes, were prepared and a nonenzymatic reduction system including two-step-reduction and process optimization was established to control the metHb level in the present study. After the first-step-reduction procedure was performed, the raw BHb, being oxidized severely, was encapsulated to form nanoparticles by the modified double emulsion method. The binary solvent of dichloromethane (DCM) and acetonitrile (Aci) showed properties such as minimizing Hb oxidation and enlarging the pores of nanopartilces. Based on the size of pores o f nanoparticles evaluated by the diffusion of various substances wi thdifferent molecular weights, reducing agents such as ascorbic acid and glutathione present in the plasma were selected to perform the second-step-reduction, i. e. to further reduce the metHb in nanoparticles. The metHb level was reduced from over 90% in the raw materials to 1.25% by the two-step-reduction and controlled preparation; this is near the level of native blood, possessing the ability of carrying/releasing oxygen.