The invention discloses an in-vitro insemination method of litopenaeus vannamei. The method comprises the following steps of ripening of litopenaeus vannamei parent shrimps, acquisition of sperms, sperm capacitation, combination of sperms and eggs and detection of the survival rate of the sperms. The method specifically comprises the following steps of ripening the litopenaeus vannamei parent shrimps, acquiring the sperms by means of a net fruiting method or a tabletting method, culturing the sperms in a capacitation solution to obtain capacitated sperms, mixing the capacitated sperms with theeggs, performing electric shock stimulation, and finally obtaining litopenaeus vannamei embryos through water bath culture. According to the method, on the basis of sperm characteristics of litopenaeus vannamei, proper capacitation liquid is selected to culture the sperms of litopenaeus vannamei in order to improve the insemination capability of the sperms; meanwhile, in the insemination process,electric shock stimulation is carried out on a sperm-egg mixed liquid, so that the sperm-egg combination of litopenaeus vannamei is promoted, and the artificial insemination effect is improved.