The invention discloses a long-time in vitro preservation method for citrus yellow vein clearing virus. The method comprises the following steps: adding an isometric protecting agent into an active citrus yellow vein clearing virus extracting solution, mixing upside down to obtain a mixture, quickly putting the mixture into liquid nitrogen for 2-3 minutes, and then transferring into an ultralow-temperature refrigerator with the temperature of -80 DEG C, wherein the protecting agent is a solution which is prepared by a PBS buffering solution and contains 13-17 percent of glucose, 5-7 percent of peptone and 10-30 percent of glycerinum; the PBS buffering solution is prepared by dissolving and sterilizing 6.8-8g of NaCl, 2.2-2.9g of Na2HPO4.12H2O, 0.1-0.2g of KH2PO4, 0.15-0.2g of KCl, 0.2-0.5g of NaN3 and 0.5-1.5mL of Tween-20 in 1,000mL of deionized water for later use. The citrus yellow vein clearing virus preserved by the method in vitro still can keep extremely high infection activity after 2 years.