A eukaryotic expression vector for the human breast cancer HYAL1 gene was constructed and stably transfected with MCF-7 and ZR-75-30 cell lines with low HYAL1 gene expression to lay the foundation for further investigation of the role of HYAL1 gene in breast cancer invasion and metastasis. Methods The hyaluronidase HYAL1 gene was amplified from a human breast cancer cell line MDA-MB-435S with high HYAL1 expression by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.1/V5-His-TOPO, and the constructed recombinant expression plasmids were transfected by liposome-mediated transfection of breast cancer MCF-7 and ZR-75-30 cells with low HYAL1 gene expression. 75-30 cells. Results A DNA fragment of 1332 bp was successfully amplified by RT-PCR, and the eukaryotic expression vector of human breast cancer HYAL1 gene was successfully constructed as demonstrated by restriction endonuclease digestion analysis and DNA sequencing.RT-PCR demonstrated that breast cancer MCF-7 and ZR-75-30 cells transfected with the recombinant plasmid could express HYAL1 gene efficiently and stably, and its The ability to penetrate the extracellular matrix was enhanced. Conclusion Eukaryotic expression vectors for human breast cancer HYAL1 gene were successfully constructed, and MCF-7 and ZR-75-30 cell clones stably expressing human HYAL1 gene with enhanced invasive ability were obtained.