The invention discloses a preparation of method of a plasmodium vivax aldolase protein monoclonal antibody. According to the antibody, aldolase protein is specifically used as targeting antigen; three dominant antigen epitopes A, B and C are selected and connected by flexible segments so as to form a recombinant D; enzyme cutting sites of BamHI and XhoI restriction enzymes are added on the upper course and the lower course of the recombinant, respectively; then the product after double enzyme cutting processes is inserted into PET-28a (+) carrier so as to structure a recombined protein D expression carrier; the recombined protein D is expressed by escherichia coli BL21; a Balb/c mouse is immunized; spleen cells of the mouse are taken and fused with sp2/0 myeloma cells; and finally ten hybridoma cell lines capable of stably secreting aldolase protein monoclonal antibody are obtained after filtering. The monoclonal antibody obtained by the method provided by the invention is capable of specifically identifying tertian plasmodium aldolase protein so that the monoclonal antibody is beneficial for the specific detection of tertian plasmodium infection; in addition, the specificity is high, the response is sensitive, and the experiment cost is low, so that the antibody is suitable for the high-pass rapid detection.