Folate receptor alpha (αFR) is overexpressed in 90% of ovarian cancers, one of the most lethal gynecologic cancers. Recent studies have suggested that natural killer (NK) cells may be better chimeric antigen receptor (CAR) drivers because of their favorable innate characteristics, such as directly recognizing and killing tumor cells. However, the therapeutic effects of CAR-engineered NK cells targeting αFR in ovarian cancer have not been reported. In this research, 3 generations of anti-αFR CAR were constructed, namely αFR-ζ (first generation), αFR-28ζ (second generation), and αFR-28BBζ (third generation), and were highly expressed on the surface of NK-92 cells by lentivirus gene transfection. Three anti-αFR CAR-engineered NK-92 cells can specifically kill αFR-positive tumor cells in vitro, especially ovarian cancer cells with high αFR expression. Compared with NK-92 cells expressing αFR-ζ or αFR-28ζ, NK-92 cells expressing αFR-28BBζ showed not only higher antigen-specific cytotoxicity and proliferation but also lower antigen-induced apoptosis. Moreover, stronger degranulation and cytokine secretion were detected in NK-92 cells expressing αFR-28BBζ cocultured with αFR-positive tumor cells. Real-time cell analysis and live cell imaging recorded the process of NK-92 cells expressing αFR-28BBζ killing ovarian cancer cells in vitro. Furthermore, NK-92 cells expressing αFR-28BBζ can effectively eliminate cancer cells in a mouse xenograft model of ovarian cancer and significantly prolong the survival of tumor-bearing mice. These results demonstrate that the anti-αFR CARs redirect NK-92 cells with specific antitumor activity, and the third-generation anti-αFR CAR-engineered NK-92 cells display more potent cytotoxicity against αFR-positive ovarian cancer, laying the foundation for future clinical research.