The invention discloses a streptococcus suis 2-type Rex gene knock-out mutant SS2-1 delta Rex and a construction method and an application thereof, belonging to the field of gene engineering. The mutant SS2-1 delta Rex is obtained by knocking out the Rex coding gene from the SS2-1 strain genome. The construction method of the mutant SS2-1 delta Rex is as follows: constructing a gene knock-out plasmid pSET4s::Rex containing homologous recombination fragment, electrotransforming the pSET4s::Rex into the SS2-1 strain, performing homologous recombination, and carrying out resistance selection, wherein the two sides of the homologous recombination fragment are forward and reverse fragments in Rex coding gene in SS2-1 strain genome, while resistance gene fragments are in the middle. Compared with the parent strain, the toxicity of the mutant strain SS2-1 delta Rex is reduced dramatically, the construction method is simple and efficient, and the infection of SS2 is effectively controlled by only two inoculation of the live vaccine of mutant strain SS2-1 delta Rex with no obvious toxic or side effects.