Low efficiency and stability problems have been major issues in the formulation of engineered monoclonal antibodies (mAbs) for a variety of therapeutic uses, which may be severer for applying to encapsulation into nanoparticle (NP). In this study, the formulation and stabilizing conditions to encapsulate a potential mAb (3D8 scFv) into biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs were investigated. And the effect of stabilizers on the stability of 3D8 scFv was investigated with the 3D8 scFv that was recovered from the primary emulsion in the double emulsion process. The conformational stability of the recovered 3D8 scFv was evaluated by circular dichroism (CD) and fluorescence spectroscopy. The DNA binding and hydrolyzing activities of the recovered 3D8 scFv were evaluated by enzyme-linked immunosorbent assay (ELISA) and agarose gel electrophoresis, respectively. The results conclude that mannitol was the most suitable stabilizer for retaining stability and activity of 3D8 scFv in the process of the PLGA NP preparation. Finally, obtained results suggest that this systematic process can provide efficient delivery system of 3D8 scFv as well as other potential mAbs or proteins for therapeutic uses, which is likely to be useful for intracellular delivery requiring sustained release.