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In order to realize long-term carrying/delivering oxygen and minimize the adverse effects of free hemoglobin (Hb) in vivo, Hb is desired to be confined in Hb-loaded nanoparticles (HbP), a novel blood substitute with potential clinical applications, and thus functions as the native red blood cells (RBCs). However, the initial burst release of Hb (“leaky effect”) greatly underscores the significance of this work. The study described here wants to disclose the key preparative parameters, including polymer, excipients in the inner aqueous phase and solvent profile, affecting the Hb release behavior (the initial 24 h) from HbP fabricated by commonly used solvent diffusion/evaporation double emulsion technique. The results demonstrate that PEGlytated polymers, regardless of two- or tri-block copolymers show slower release compared with the corresponding non-PEGlytated ones. The higher polymer concentration yields lower initial release. PEG200, added as excipient facilitates Hb burst effect to about 38.4%, almost 17% increase compared to the control (∼21%), whereas, PVA and Poloxamer188, due to amphiphilic nature, can effectively attenuate this leakage to about 13.0 and 5.1%, respectively. The diffusion/extraction rate from oil phase and the subsequent evaporation rate from the aqueous continuous phase of solvents impose different influences on Hb release. To reduce the burst effect, the initial diffusion/extraction rate should be slow, whereas, the concomitant evaporation rate should be as fast as possible. The results obtained here will be guidance's for the future tailored design of more desirable polymersome nanoparticle blood substitutes.