Two distinct preparations of amphiphilic diblock copolymer vesicles (i.e., polymersomes), composed of (poly(ethylene oxide)-poly(butadiene)) (PEO-PBD), with molecular weights of 1.8 and 10.4 kDa, offering different hydrophobic membrane thicknesses, were used to encapsulate the oxygen (O-2) storage and transport protein hemoglobin (Hb) for possible application as a red blood cell (RBC) substitute. Key biophysical properties as well as the kinetics of polymersome encapsulated Hb (PEH) interaction with physiologically important gaseous ligands (O-2, carbon monoxide, and nitric oxide) were measured as a function of the hydrophobic membrane thickness of the PEH particle. Taken together, the results of this work show that PEHs exhibit biophysical properties and retarded ligand binding/release kinetics (compared to cell-free Hb), which are similar to the behavior of RBCs. Therefore, PEHs have the potential to serve as safe and efficacious RBC substitutes for use in transfusion medicine.