Chemokines are known to stimulate directed migration of cancer cells. Therefore, the strategy involving gradual chemokine release from polymeric vehicles for trapping cancer cells is of interest. In this work, the chemokine stromal cell-derived factor-1α (SDF-1α) was encapsulated into nanoparticles composed of poly-(lactic-co-glycolic acid) (PLGA) and a polyethylene glycol (PEG)-PLGA co-polymer to achieve sustained release. SDF-1α, and lysozyme as a model protein, were firstly precipitated to promote their stability upon encapsulation. A novel phase separation method utilising a non-toxic solvent in the form of isosorbide dimethyl ether was developed for the individual encapsulation of SDF-1α and lysozyme precipitates. Uniform nanoparticles of 200–250 nm in size with spherical morphologies were successfully synthesised under mild formulation conditions and conveniently freeze-dried in the presence of hydroxypropyl-β-cyclodextrin as a stabiliser. The effect of PLGA carboxylic acid terminal capping on protein encapsulation efficiency and release rate was also explored. Following optimisation, sustained release of SDF-1α was achieved over a period of 72 h. Importantly, the novel encapsulation process was found to induce negligible protein denaturation. The obtained SDF-1α nanocarriers may be subsequently incorporated within a hydrogel or other scaffolds to establish a chemokine concentration gradient for the trapping of glioblastoma cells.