Through adding cross-linking agent into water-in-oil (W/O) emulsion system of Lipase solution, Lipase spherezymes is formed after emulsifying cross-linked by centrifugation. This paper studies different cross-linking agents, different cross-linking time and different enzyme mass concentration to the affect of spherezymes activity. This paper concludes that the suitable condition of making Lipase spherezymes is: 25% glutaraldehyde solution and 3% polyethyleneimine solution are allowed to react for 5min in a 1︰1 volumetric ratio at room temperature, then the Glu-PEI copolymer is formed and used for cross-linking agent of Lipase spherezymes, and cross-linking time is 2 hour and enzyme concentration is 50g/L, the residual ratio of the Lipase spherezymes activity is up to 86.3%.