To improve the content of umami substance in boiling water of Lentinus edodes, the single factor experiment and orthogonal experiment were carried out to optimize the enzymatic hydrolysis process of L. edodes boiling water with papain. The amino acid nitrogen content and equivalent umami concentration (EUC) were also evaluated after enzymatic hydrolysis by papain. Results showed that the optimum conditions for enzymatic hydrolysis to improve the content of amino acid nitrogen in boiling water of L. edodes by papain were as follows: pH value 6.0, enzymatic temperature 55 ℃, enzymatic amount 0.4% and enzymatic hydrolysis time 5 h. Under the control of these conditions, the content of amino acid nitrogen was 0.454 g/100 mL. Enzymatic hydrolysis could significantly increase the contents of glutamic acid and aspartic acid in boiling water of L. edodes as well as EUC.