[Objective] To examine the effects of metal ions and protectants on activity of myrosinase and explore purification method of myrosinase produced by Trichederma atroviride T155. [Methods] By concentration of metal ions and protectants to study of the myrosinase activity was measured by adding different ions with different concentration and three protectants, respectively. The pure myrosinase was obtained by cell disruption, ammonium sulfate precipitation, dialysis, Sephadex G-100 chromatography, DEAE-52 chromatography and Sephadex G-200 chromatography in order. [Results] The results showed that Ag+, Zn2+, Pb2+, Mg2+, Hg2+, Fe3+ promoted enzyme activity of myrosinase at low concentration and inhibited enzyme activity at high concentration, Ca2+ promoted enzyme activity at the concentration of 0.069-17.000 g/L. 1 mmol/L EDTA associated with 3 mmol/L DTT was the best treatment for keeping myrosinase activity, and 85% enzyme activity was retained after 27 days of preservation at 4 °C. Molecular weight of the myrosinase produced by T155 was about 150 kD by SDS-PAGE, and this myrosinase was a dimer. [Conclusion] The tested metal ions inhibited myrosinase activity except for Ca2+, EDTA or associated with DTT showed preferable effect on maintaining myrosinase activity. A dimer myrosinase was obtained by separation and purification technology. This study intended to provide a new way and idea for mining myrosinase producing microorganism.