The microcosmic mechanisms of protein (recombinant human growth hormone, rhGH) incomplete release and stability from amphiphilic poly(monomethoxypolyethylene glycol-co-D,L-lactide) (mPEG-PLA, PELA) microspheres were investigated. PELA with different hydrophilicities (PELA-1, PELA-2, and PELA-3) based on various ratios of mPEG to PLA were employed to prepare microspheres exhibiting a narrow size distribution using a combined double emulsion and premix membrane emulsification method. The morphology, rhGH encapsulation efficiency, in vitro release profile, and rhGH stability of PELA microspheres during the release were characterized and compared in detail. It was found that increasing amounts of PLA enhanced the encapsulation efficiency of PELA microspheres but reduced both the release rate of rhGH and its stability. Contact angle, atomic force microscope (AFM), and quartz crystal microbalance with dissipation (QCM-D) techniques were first combined to elucidate the microcosmic mechanism of incomplete release by measuring the hydrophilicity of the PELA film and its interaction with rhGH. In addition, the pH change within the microsphere microenvironment was monitored by confocal laser scanning microscopy (CLSM) employing a pH-sensitive dye, which clarified the stability of rhGH during the release. These results suggested that PELA hydrophilicity played an important role in rhGH incomplete release and stability. Thus, the selection of suitable hydrophilic polymers with adequate PEG lengths is critical in the preparation of optimum protein drug sustained release systems. This present work is a first report elucidating the microcosmic mechanisms responsible for rhGH stability and its interaction with the microspheres. Importantly, this research demonstrated the application of promising new experimental methods in investigating the interaction between biomaterials and biomacromolecules, thus opening up a range of exciting potential applications in the biomedical field including drug delivery and tissue regeneration.