The present invention discloses a method of preparing 2-deoxyribose 5-phosphate by reacting glyceraldehyde 3-phosphate and acetaldehyde in the presence of either a microorganism itself which contains 2-deoxyribose-5-phosphate aldolase but substantially no phosphatase or the enzyme derived from the microorganism. The present invention also discloses a method of preparing 2-deoxyribose 5-phosphate by reacting dihydroxyacetone phosphate and acetaldehyde in the presence of either a microorganism itself which contains 2-deoxyribose-5-phosphate aldolase and triose-phosphate isomerase but substantially no phosphatase or the enzymes derived from the microorganism.