Hb (hemoglobin)-loaded particles (HbP) encapsulated by a biodegradable polymer used as oxygen carrier were prepared. A modified double emulsion and solvent diffusion/evaporation method was adopted. All experiments were performed based on two types of biodegradable polymers, poly(epsilon-caprolactone) (PCL) and poly(epsilon-caprolactone-ethylene glycol) (PCL-PEG). The biodistribution and the survival time in blood of the particles were investigated in a mouse model. Encapsulation efficiency and pore-connecting efficiency were evaluated by a novel sulfocyanate potassium method. The influence of process parameters on the particle size and pore-connecting efficiency (PCE%) of nanoparticles have been discussed. The prepared conditions: solvent, external aqueous phase, pressure were discussed. The system utilizing dichloromethane (DCM)/ethyl acetate (EA) as a solvent with an unsaturated external aqueous phase yielded the highest encapsulation efficiency (87.35%) with a small mean particle size (153 nm). The formation of porous channels was attributed to the diffusion of solvent. The PCE% was more sensitive to the rate of solvent diffusion that was obviously affected by the preparation temperature. The PCE% reached 87.47% when PCL-PEG was employed at 25 degrees C. P(50) of HbP was 27 mmHg, which does not seem to be greatly affected by the encapsulation procedure. In vivo, following intravenous injection of 6-coumarin labeled HbP, the major organ accumulating Hb-loaded particles was the liver. The half-life of nano-sized PCL HbP was 3.1 times as long as the micro-sized PCL HbP. Also, Nano-sized as well as a PEGylated surface on HbP is beneficial for prolonged blood residence (7.2 fold increase).