The invention discloses a preparation method of inonotus obliquus non sugar extractives and application thereof. The preparation method comprises the steps of culturing and fermenting inonotus obliquus, performing cold induction treatment on a culture solution, preparing freeze-drying powder, performing chloroform extraction on the freeze-drying powder to obtain a preliminary extracting solution,then, dialysis treatment is performed, finally, high performance liquid chromatography is performed, and purification is performed to obtain a refined extractive. The preparation method is simple andshort in period, the main ingredients of the prepared non sugar extractives are triazine derivatives, the good bacteriostat effect is achieved for bacteria and fungi, the extractives are biological preparation, little environment contamination is generated, and the method can be applied to preparing environment-friendly bactericide and bacteriostat medicine, the good application prospect is achieved, the application value for inonotus obliquus is sufficiently developed, and the utilization rate for medicinal fungi is increased.