Lys-N, also known as lysine-specific metalloendopeptidase, functions as the "sister" enzyme of lysyl endopeptidase (Lys-C) in proteomic research. Its digestion specificity at the N-terminal lysine residue makes it a very useful tool in proteomics analysis, especially in mass spectrometry-based de novo sequencing of proteins. Here we invented a complete production process of highly purified Lys-N from dry fruit of Grifola frondosa (Maitake Mushroom). The purification process includes 1 step of microfiltration plus 1 step of UF/DF (ultra-filtrated used in tandem with diafiltrated method) recovery step and 4 steps of chromatography purification. The overall yield of the process was approximately 6.7 mg Lys-N protein/kg dry fruit of Grifola frondosa. The assay data demonstrated that the purified Lys-N exhibited high enzymatic activity and specificity. The novel production process provides the first time to extract of Lys-N from dry fruit of Grifola frondosa. The process is also stable, scalable, and provides an economic way of producing the enzyme in large quantities for mass spectrum-based proteomics and other biological studies.