1,3-dihydroxyacetone (DHA) is one of the most valuable chemicals with a wide range of applications in cosmetics and pharmaceutical industry. Glycerol dehydrogenase (GlyDH) is able to selectively catalyze the transformation of glycerol to DHA, by which glycerol, the oversupply by-product of the biodiesel industry is highly valued. To make enzymatic production of DHA practically feasible, immobilizing GlyDH onto a suitable insoluble support is critical for facilitating biocatalyst recovery and improving stability of the enzyme. Agarose microspheres as carrier were prepared by membrane emulsification method. Then the agarose microspheres were activated with epichlorohydrin and reacted with polyethyleneimine (PEI) to introduce the amino group into the agarose microspheres. The morphology and structure of the microspheres were characterized by scanning electron microscope (SEM), energy dispersion spectrum (EDS), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR). GlyDH was covalently fixed with glutaraldehyde and then catalyzed the formation of 1,3-dihydroxyacetone. The results showed that the spherical degree of microspheres was enhanced due to the membrane emulsification process. The N content in grafted products was obviously effected by the relative molecular weight of PEI. The N content of PEI graft was the highest when the relative molecular mass of PEI was 10000. The effect of pH and temperature dependence, thermal stability and the reusability of GlyDH were systematically investigated. The optimum pH value of free enzyme and immobilized enzyme activity was 10.0. The storage efficiency of immobilized GlyDH was higher than that of free GlyDH after storage at 4℃ for 21 days. After 7 cycles, the activity of GlyDH was still partly remained.