This invetion relates to Pullulan membrane and its use for culture preservation. The said membrane is prepared by flow spreading process to form thickness 0.02-0.04mm, pull strength 27-32MPa, right tearing strength 31-38KN/m, light transmittance rate 95-98%, and oxygen penetration (2.7-1.6cm3/m2.bar.d). The method is carried out by: providing glycerin, trehalose as protecting agent and microbe between both of membranes, pressing tightly and sealing with plastic layers to meet conditions for culture preservation, e.g., dryness, oxygen depletion, low temperature, nutrient depletion and protecting agent addition. Those sheets can be formed into albums for preservation, research and easy reproduction by cutting a piece and putting into a culture dish. No expensive apparatus and large space for preservation are needed and it is easily posted.