The invention provides a preparation method of high-potency positive serum for a porcine pseudorabies virus. The method takes a rabbit as an immunization object, and immunizes the rabbit with an inactivated vaccine containing a suitable amount of PolyI:C, muramyl dipeptide and cimetidine to produce the hyperimmune serum, can achieve the effect that no cytopathic effect occurs in subculture of at least 2 generations in a T25 square flask equal volume ratio neutralization 1 vaccine experiment, and meets the requirement that a cell method serves as an exogenous virus detection standard in the (2015 edition). The preparation method of the high-potency positive serum for the porcine pseudorabies virus has a good application prospect.