Beta-1,4-mannotriose was prepared by the enzymatic hydrolysis of poonac and the subsequent elimination with yeast of monosaccharides and disaccharide from the resultant hydrolysate. The enzyme system hydrolyzed poonac and produced monosaccharides, disaccharide and ${eta}$-1,4-mannotriose without other oligomers at the final reaction stage. Poonac (50 g) was hydrolyzed at $50^{circ}C$ and pH 6 for 48 hr with the crude enzyme solution (500 mL) from Trichoderma harzianum. The elimination of monosaccharides and disaccharide from the hydrolysis products with a yeast (Candida guilliermondii) produced 10.5 g of crystalline [${eta}$-1,4-mannotriose without the use of chromatographic techniques. After 48 hr of yeast cultivation, the total sugar content fell from 4.8% to 3.4%, and the average degree of polymerization (D.P) rose from 2.5 to 3.2. The preparation method presented was confirmed to be suitable for the preparation of mannotriose from poonac.