The invention discloses a purification method of conotoxin, and mainly solves the technical problem of low recovery rate of the existing purification methods. The technical scheme is as follows: a purification method of conotoxin, comprising the following steps of (1) adding purified water to a conotoxin sample, stirring under ultrasonic waves, adjusting the pH to be alkaline, performing ultrasonic stirring and dissolving until clarification, and filtering with a microporous membrane for later use; (2) adding the sample to a reverse phase chromatography system filled with polymer filler, wherein the phase A of an eluted mobile phase is HAC with the mass percentage concentration of 0.2 to 0.3%, and the phase B of the mobile phase is acetonitrile; (3) setting elution gradient, flow rate anddetection wavelength; (4) collecting a target fraction, decompressing and performing rotary evaporation for later use; (5) performing secondary purification on the fraction by using a reverse phase high performance liquid chromatography, wherein the phase A of a mobile phase is TFA with the mass percentage concentration of 0.1%, and the phase B of the mobile phase is acetonitrile; (6) collecting atarget fraction, decompressing, performing rotary evaporation and freeze-drying.