Abstract: To find out the best explants and the best disinfection combination for initiation culture, three types of explants, namely, young leaves, moderately mature leaves and petioles, were selected and tested orthogonally with 75% ethanol+2% sodium hypochlorite and 20% hydrogen peroxide+2% sodium hypochlorite respectively in two groups of orthogonal experiments. The results show that the type of explants was the most critical factor affecting the disinfection effectiveness. The moderately mature leaves, which had just turned from fresh green to green, are the best explants with the lowest contamination rate (7.4%) and the highest callus induction rate (88.9%). The young leaves are easily injured and browned when disinfected, and the induction rate is less than that of moderately mature leaves but higher than that of petioles. The petioles have the highest contamination rate (41.7%), slow callus inducting and less callus quantity; In general, the disinfection and induction effect of leaf was better than that of petiole. The best disinfection effect could be achieved by combining 2% sodium hypochlorite with 75% ethanol, which is the preferred agent for the disinfection of Pulsatilla chinensis leaves. When 20% hydrogen peroxide is used for disinfection, the browning of the explant material was serious. The best combination for the disinfection of Pulsatilla chinensis from Henan Dabie mountain is moderately mature leaves+75% ethanol for 20 s+2% sodium hypochlorite for 8 min, or moderately mature leaves+75% ethanol for 20 s+2% sodium hypochlorite for 12 min.