Poly(3-hydroxybutyrate) (PHB) based microspheres were prepared via double emulsion solvent evaporation using polyethylene glycol (PEG), poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), polylactide (PLA), poly(dl-lactic-co-glycolic acid) (PLGA) or chitosan (CTS) as the additive of wall polymers. It was found that additives had distinct effect on the properties of microspheres, such as the yield, drug loading, average diameter, crystallization states microstructure and surface morphological characters. PHB based microspheres using PEG as the additive had the lowest yield, the smallest average diameter, and the highest drug loading which reached 12.2% thereinto. At the same time it had the lowest crystallinity of PHB, and the diameter of the crystal particles was only 11.44 nm. It was feasible to prepare PHB based microspheres using PEG and PHBV as additives, which had relatively high protein loading but different microstructures and surface morphologies, and they were anticipated to have a good effect of controlled release.