Increasing researches have focused on cancer metastasis and development. The ectonucleotidase CD73 is one of the most common cell surface enzymes that are involved in immunosuppression. In this study, the recombinant plasmid pET28a-CD73 was constructed and the CD73 protein was overexpressed in as an inclusion body that was then subjected to refolding. The anti-CD73 monoclonal antibody (3F7) was obtained by hybridoma technology. The antibody subtype was identified as IgG2a with an affinity constant of 5.75 nM. This antibody could be applied to immunofluorescence and flow cytometry. The results showed that the CD73 protein was not only located in the cytoplasm but also distributed on the surface of triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468. Moreover, the level of CD73 protein was associated with the survival rate. Although the anti-CD73 antibody was not able to inhibit tumor cell growth, it could enhance the cytotoxic effect of Doxorubicin to triple-negative breast cancer cells. In vitro function assay results indicated that anti-CD73 mAb could inhibit cell migration and invasion in both human triple-negative breast cancer and mouse 4T1 cell lines. In this process, both the LC3I/LC3II ratio and p62 protein levels increased, which indicated that the blockage of CD73 could inhibit cell autophagy, and cell migration and invasion were restored by rapamycin. In vivo, anti-CD73 mAb could significantly inhibit lung metastasis of 4T1 cells in a mouse xenograft model. Taken together, this novel anti-CD73 antibody could be developed as an adjuvant drug for triple-negative breast cancer therapy and can be useful in tumor diagnosis.