The invention discloses a black pleurotus ostreatus protein PoAP and a preparation method thereof. The preparation method comprises the following steps: firstly, lixiviating black pleurotus ostreatus hypha dry powder in phosphate buffer solution for 1-3 hours and then centrifugating to collect supernatant liquor; then adding ammonium sulfate to the collected supernatant liquor to a saturation of 40 percent and a saturation of 70 percent respectively, standing at 2-6 DEG C for 6-12 hours; and centrifugating to collect precipitation; using phosphate buffer solution to dissolve precipitation andthen utilizing distilled water to dialyze; then separating through an anion exchange column, and eluting by 0.5 m NaCl and collecting elution peak; and then ultra-filtrating at the speed of 5,000 rpmfor 40 minutes through a 30KD ultra-filtration pipe, then obtaining the black pleurotus ostreatus protein which is the material in the obtained solution. The black pleurotus ostreatus protein PoAP issingle protein which can be applied on genetic engineering; and the invention has good inhibitory effect on tobacco mosaic virus. As the black pleurotus ostreatus protein PoAP is natural extract, theinvention is harmless to humans and environments. In addition, the preparation method of the black pleurotus ostreatus protein is simple and has the advantages of easily-obtained raw material and lower production cost.