The invention provides active UL36-480-GST protein as well as a preparation method. The preparation method comprises the following steps: with the MDV virus coded UL36-480 deubiquitinating enzyme as atarget, fusing the C end of the target with a GST label promoting protein dissolution to facilitate purification, a Thrombin protease digestion site and a Gly-gly-Gly-Ser flexible connection peptidefragment; expressing and purifying with a rhabdovirus-insect cell expression system to obtain UL36-480-GST. A specific inhibitor and a method for anti-tumor and anti-virus treatment are screened in vitro by use of the enzyme; a new preparation method for obtaining active virus coded deubiquitinating enzyme UL36-480-GST is established, and a new specific drug target is provided for effectively preventing and controlling MDV viruses.