The paper studied preparation of L-rhamnose by two-steps biotransformation of naringin using naringinase and rest cells of yeast as catalyst. The optimum conditions of hydrolysis was the concentration of naringin at 14g/L, 10mL/L of naringinase, pH at 5.5, reaction time of 22h. The optimum conditions of fermentation was the yeast dosage 2.5g/L, the temperature at 32 ℃, pH at 6.0, fermentation time of 120min. A small-scale pilot was carried out in 30L fermentor, 85.6g crystals of rhamnose with more than 98.5% purity was get. The result was the theoretical yield of 86.5%.