Lysozyme-encapsulated chitosan microspheres with micron-size diameters were successfully prepared for the first time by employing the Shirasu porous glass (SPG) membrane emulsification technique followed by cross-linking with glutaraldehyde, and the relationships between the preparation conditions and characteristics of the microspheres were studied in detail. This preparation method provided size-controllability and monodispersity of the microspheres, owing to the sharpness of the pore sizes of the SPG membranes. It was also possible to predict the average diameters of the enzyme-encapsulated microspheres using no fitting parameters, on the basis that each microsphere is prepared in an emulsion containing chitosan and lysozyme, without any collisions or aggregation occurring. X-ray photoelectron spectroscopy measurements indicated that the amount of encapsulated lysozyme was controlled by the concentrations of chitosan and lysozymes in the dispersion phase used for preparing the emulsions from which the enzyme-encapsulated microspheres are formed. Finally, the apparent activity of the encapsulated lysozymes was measured by the viscosimetric method, using ethyleneglycolchitin. Results showed that about half of the activity of the encapsulated lysozymes was maintained during the preparation procedure when employing the SPG membrane emulsification technique.