Hollow polyurethane (PU) microcapsules were prepared by co-axial electrospraying technology, with glycerol containing certain content of water and PU dissolved in N, N-dimethylacetamide (DMAC) as internal and external phase electro-spraying solutions, respectively. The effects of the solution composition and flow rate on the morphology of the microcapsule were investigated. The results showed that uniform hollow PU microcapsules were successfully fabricated, using co-axial electrospraying of 80%(?) glycerol and DMAC solution of 10%(?) PU as internal and external phase electrospraying solution, under the conditions of the solution flow rates of 0.05 and 1 mL/h, voltage about 22 kV, distance of 48 cm between spinneret and collector, temperature of 30℃, and humility of 10%. By dissolving definite amount of Candida antarctica lipase B (CALB) in the internal phase electrospraying solution, the enzyme was in-situ encapsulated inside the hollow cavity of PU microcapsules. The original biocatalytic activity of encapsulated CALB was 76.19% of the free CALB, and about 60% after 10 times recycles. Thermal stability of CALB showed that the half-life of CALB at 60℃ was prolonged about 8 times after encapsulation.