Abstract: Virus-free in vitro bulblets rapid propagation system was established by optimizing the medium accretion and transplantation matrix of the bulbs after heat treatment using Lilium Longiflorum bulbs with virus as experimental material. The results showed that the regeneration rate was up to more than 86% when added MS with phytohormone NAA and KT which can promote the bulblets formation. The best bulblets formation was observed in the medium with 0.4mg?L-1 KT and 0.2mg?L-1 NAA. Optimum bulblet enlargement was observed in medium with Paclobutrazol 5mg/L, while Paclobutrazol in concentration 3mg?L-1 was propitious to bulblet formation. The bulblets enlargement can not be affected significantly by AC, but the quality of bulblets can be enhanced in the medium with appropriate concentration of AC. Medium with higher concentration sucrose can promote bulblet enlargement but inhibit bulblet formation. The best matrix for the highest surviving rate and best growth situation was pearlite/soil (3:1). The number of bulb scales surviving and regenerating decreased significantly as the increasing of days after the treatment of high temperature 37oC. The regeneration rate can be about 40% for 20days. The effectiveness of eliminating LSV with the method got to more than 95% which was significantly higher compared with the control.