Abstract: A multi-step membrane separation process whereby the natural polymer .alpha.-chitosan extracted from the shell of crustaceans, etc. can be simply separated and purified into low molecular weight chitosan or oligosaccharide chitosan in a high yield. A chitosan mixture of various molecular weights is reacted with an acid in the presence of enzyme while applying an ultrasonic treatment to the mixture and then, added with pure water to give an aqueous solution having a chitosan concentration of 1 to 50%. It is subjected to a continuous membrane filtration under a pressure and further to a second fractionation and separation technique employing a proper membrane according to a desired molecular weight cut-off, at least one time.