A new method for hemoglobin (Hb) deoxygenation and re-oxygenation, in suspension or within red blood cells, RBCs, is described using the commercial enzyme product, EC-Oxyrase®. This method using EC-Oxyrase has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin, eliminating the need of a sparging deoxygenation gas and airtight vessels, as well as easy re-oxygenation by washing and adding to a normal buffer with dissolved oxygen (DO). Spectra of deoxyHb and metHb from RBCs using three preparation methods: sodium dithionite, sodium nitrite and Oxyrase, show high purity of the deoxyHb using Oxyrase (with little to no methemoglobin or hemichrome production from side reactions). The deoxygenation action of Oxyrase follows first order reaction kinetics. Paramagnetic characteristics of intracellular hemoglobin in RBCs are compared using cell tracking velocimetry for healthy and sickle cell disease (SCD) donors and oxygen dissociation curves show that the function of healthy RBCs is unchanged after Oxyrase treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyhemoglobin, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to the existing methods.