Venom of Agkistrodon halys Pallas can controlsevere pain such as cancer pain and neuropathic pain, but itis made up of complicated components. Aim of this studyis to separate major analgesic fraction from venom ofA. halys Pallas, and to reveal its biochemical and pharmacologicalproperties. Three steps with ion exchangecolumn first and molecular sieve columns next were usedto separate and purify the fractions of venom. Analgesiceffects were evaluated by hot plate tests and writhing testsin mice. The molecular weight (MW), isoelectric point,amino acid sequence, purity were respectively determinedby SDS-PAGE electrophoresis, isoelectric focusing,Edman degradation and HPLC. The dependence and tolerancewere observed by withdrawal test in rats, andanalgesic effects were observed in mice during 7 daysadministration. Fourteen fractions were obtained by separation;the best analgesic fraction named Pallanalgesinwas selected by ED50 and LD50. It had single band inelectrophoresis, relative purity 92.16 %, MW 16.6 kDa,isoelectric point 8.8, and former sequence of ten aminoacids H-L-L-Q-F-R-K-M-I-K. It showed significant analgesiceffect without tolerance and dependence. As a novelanalgesic, Pallanalgesin has been found to explain thefunction of venom of A. halys Pallas on severe pain controlin traditional uses.