The aim of this study was to incorporate human recombinant erythropoietin (EPO) in biodegradable polymeric nanoparticles targeting a prolonged-release effect. EPO-loaded poly(DL-lactide-co-glycolide) nanoparticles were prepared using double emulsion method (w/o/w) with least process-related stress on the encapsulated drug. The nanoparticles have been fully characterized including in vitro release profile. The biological activity was assessed in vivo using BALB-c mice. The produced particles appeared spherical in shape with smooth regular surfaces and had an average particle size of 225.9 ± 3.8 nm. The entrapment efficiency was 33.3%. The in vitro release profile exhibited a biphasic mode with a burst of 50% cumulative drug release, followed by a slow rate of release over 24 h, reaching a maximum of 82%. The bioassay results showed that EPO-loaded nanoparticles were able to maintain the physiological activity of EPO for 14 days after single subcutaneous injection compared with pure and marketed EPO formulae (EPREX®).