This study investigates a method of preparing hazard-resistant protein-loaded polysaccharide glassy microparticles using freezing-induced phase separation method without exposure to water/oil, water/air interface and cross-linking reagents. Model protein (such as bovine serum albumin, myoglobin and beta-galactosidase (beta-Gal)) was dissolved in water together with dextran and polyethylene glycol (PEG), followed by a freezing process to form a temperature-stabilized aqueous-aqueous emulsion wherein dextran separated out as the dispersed phase with protein partitioned in preferentially. The frozen sample was freeze-dried and washed with dichloromethane (DCM) to remove the PEG continuous phase, after which protein-loaded polysaccharide particles, 1-4 microm in diameter, were harvested. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) patterns showed that the particles were in glassy state. These glassy polysaccharide microparticles can well protect the delicate structure of proteins and preserve their bioactivities under deleterious environment interacting with organic solvents, vortex and centrifugation processes that often involve during the formulation processes leading to polymer-based sustained-release systems. Therefore, this freezing-induced phase separation method is a mild and effective way to encapsulate protein into hazard-resistant polysaccharide glassy particles, which ensure its stability in subsequent formulating processes that leads to polymer-based sustained-release system.