Abstract: In this study, the pyrF screening marker and the genomic DNA fragments were used to construct the expression vectors in food-grade Lactococcus lactis (L. lactis). Such expression system could potentially be used to express and produce food-grade and medicinal polypeptides. Firstly, the NZ3900 ΔpyrF auxotrophic strain was created from the homologous recombination mutant cassette. Secondly, the repA and repC genes were used as the replication elements, the pyrF gene as the screening marker, the P₃₂ and P₈ elements from L. lactis as the promoters, and the Tusp₄₅ and TpepN from L. lactis as the terminators, all of which were constructed in the expression plasmid pLD. Finally, the reporter gene ZsGreen (a fluorescent protein) was used to verify the expression of recombinant protein in the NZ3900 ΔpyrF mutant strain and the genetic stability of pLD-ZsG plasmid. The result showed that the prototrophic ZsGreen positive transformants could grow normally in common Elliker culture medium, and the green fluorescent signal was observed under a fluorescence microscope. In addition, ZsGreen protein could be highly expressed in NZ3900 ΔpyrF and the expression plasmid could be stably transmitted through at least 30 generations, according to the results of the PCR and Western blotting, indicating that the recombinant protein was expressed in L. lactis in a stable manner. Based on the above results, the approach for creating an L. lactis expression vector (without antibiotic resistance gene) based on the pyrF auxotrophic marker is feasible and offers a basis for further investigation into the use of L. lactis to manufacture food- and pharmaceutical-grade polypeptides.