Novel chitosan-carrageenan nanoparticles were produced through the process of ionotropic gelation for the encapsulation and controlled release of recombinant human erythropoietin (rHu-EPO). The effects of chitosan concentration, chitosan to carrageenan mass ratio and solution pH on the nanoparticle diameter, polydispersity and surface charge were explored through both screening and response surface modeling (RSM) methods. The chitosan-carrageenan nanoparticles created had particle diameters between 200 and 1000 nm, surfaces charges between 40 and 55 mV, and polydispersity between 0.2 and 0.35. RSM optimized chitosan-carrageenan nanoparticles demonstrated an increased rHu-EPO encapsulation efficiency of 47.97 ± 4.10% and a more sustained in vitro release of ~50% over a 2 week period when compared to previous nano/microparticle delivery systems. Studies on the effect of surface charge and chitosan molecular weight on the encapsulation and controlled release of rHu-EPO revealed that increasing either led to improved encapsulation efficiency and reduced release rate.