The invention discloses novel VLR specific antibody serum as well as a preparation method and application thereof. The preparation method comprises the following steps: (S1) obtaining an artificial antigen, Gram-negative bacteria or LPS for immunization, and preparing 0.8-2mu g/mu l antigen solution from an antigen diluent; (S2) first immunization: immunizing lampetrs japonica with 90-110mu g of antigen for each; (S3) enhanced immunization: after 9-11 days of first immunization, performing intraperitoneal injection of each lampetrs japonica by using 40-60mu g of antigen every 9-11 days, wherein the enhanced immunization is performed for 2-3 times; and (S4) antiserum preparation: after 4-6 days of last immunization, cutting the tail fin and collecting blood, letting the fresh blood stay for 30-60min at 0-8 DEG C, centrifuging for 8-15min at 4 DEG C at a speed of 5,000-8,000rpm, and collecting the serum. Compared with traditional antiserum, the novel VLR specific antibody serum has stronger antigen killing ability.