The control of size and size distribution of microspheres is necessary for obtaining repeatable controlled release behavior. The chitosan microspheres were prepared by a membrane emulsification technique in this study. Chitosan was dissolved in 1 wt.% aqueous acetic acid containing 0.9 wt.% sodium chloride, which was used as a water phase. A mixture of liquid paraffin and petroleum ether 7:5 (v/v) containing PO-500 emulsifier was used as an oil phase. The water phase was permeated through the uniform pores of a porous glass membrane into the oil phase by the pressure of nitrogen gas to form W/O emulsion. Then GST (Glutaraldehyde Saturated Toluene) as crosslinking agent was slowly dropped into the W/O emulsion to solidify the chitosan droplets. The preparation condition for obtaining uniform-sized microspheres was optimized. The microspheres with different size were prepared by using the membranes with different pore size, and there was a linear relationship between the diameter of microspheres and pore size of the membranes when the microspheres were in the range of micron size. The smallest chitosan microspheres obtained was 0.4 mum in diameter. This is the first report for preparing the uniform-sized chitosan microspheres by membrane emulsification technique. Uniform chitosan microspheres were further used as a carrier of protein drug. Bovine serum albumin (BSA) as a model drug was loaded in the microspheres and released in vitro. The effects of pH value, diameter and crosslinking degree of microspheres, and BSA concentration on loading efficiency and release behavior were discussed.