The invention discloses a lactobacillus gene engineering subunit vaccine strain capable of stably expressing porcine rotavirus VP4 protein and a preparation method of the lactobacillus gene engineering subunit vaccine strain. The vaccine strain is obtained through the following steps: on the basis of lactobacillus casei with uracil phosphoribosyl transferase, UPP gene deleted, the porcine rotavirus VP4 gene is inserted between a termination codon and a terminator of the neuronspecificenoluse gene of lactobacillus casei through homologous recombination, and the vaccine strain is not provided with an antibiotic selection marker. The experiment proves that after the constructed lactobacillus gene engineering subunit vaccine strain capable of stably expressing the porcine rotavirus VP4 proteinis used for immunizing animals through oral administration, the local mucosal immune response can be induced, the mucous membrane antibody IgA is generated, the body is induced to generate humoral immune response, the serum antibody IgG is then generated, good immunogenicity is displayed, and the constructed lactobacillus gene engineering vaccine strain not labelled by antibiotic resistance accords with the development concept of 'no pollution and environmental protection' of veterinary vaccines.