The invention discloses a preparation method of a human nerve growth factor transgenic pig. The method comprises the following steps: a, constructing a CRISPR/Cas9 system expression vector of a targeted pNGF gene exon, b, constructing a homologous recombinant vector for providing an hNGF gene, c, co-transfecting porcine ear fibroblasts with the CRISPR/Cas9 system expression vector and the homologous recombinant vector of the hNGF gene, and d, screening the transfected porcine ear fibroblasts to obtain a positive cell line, and cloning somatic cells to obtain the transgenic pig with the human nerve growth factor. According to the invention, by a fixed-point gene recombination mode, the hNGF gene is insert into a pig pNGF gene locus, therefore, the obtained transgenic pig with the human nerve growth factor can express the human nerve growth factor without expressing the pig nerve growth factor, so that the current situation that when the human NGF is collected in pig saliva, the human NGF is mixed in the pig saliva is avoided, the problem of immunogenicity when the pig is used for drug therapy is also avoided, and the transgenic pig is safer and more effective.