To improve the yield of 5′-nucleotides and increase the economic value of shiitake mushroom stipe, 5′-nucleotides were extracted from shiitake mushroom stipe by cellulase. The effects of cellulase amount, liquid-solid ratio, enzymatic hydrolysis temperature, enzymatic hydrolysis time and pH on the yield of 5′-nucleotides were investigated by single factor experiments, and the orthogonal test was used to optimize the parameters. The 5′-nucleotides composition was also analyzed by high performance liquid chromatography (HPLC). Results showed that on the basis of single factor experiments, the parameters of cellulase extraction on 5′-nucleotides optimized by orthogonal test were as follows:liquid-solid ratio 20∶1 (mL∶g), cellulase amount 0.8%, enzymatic hydrolysis temperature 40℃, enzymatic hydrolysis time 4h, and pH 5.4. Under these optimum conditions, the yield of 5′-nucleotides was 4.08mg·g-1. This result was close to the content (4.57mg·g-1) measured by HPLC. The 5′-nucleotides extracted from shiitake mushroom stipe using cellulase were composed by 5′-guanosine monophosphate, 5′-uridine monophosphate, 5′-cytosine monophosphate, 5′-adenosine monophosphate, 5′-inosine monophosphate, 5′-xanthosine monophosphate, and umami taste nucleotides accounted for 62.63% of 5′-nucleotides.