Recent evidence indicates that ovarian cancer stem cells (CSCs) are responsible for ovarian cancer recurrence and drug resistance, resulting in the low long-term survival rate of patients with advanced ovarian cancer. We aimed to study the inhibitory effect of theaflavin-3, 3'-digallate (TF3), a black tea polyphenol on ovarian CSCs. Here, we showed that TF3 inhibited the proliferation of A2780/CP70 and OVCAR3 tumorshpere cells by suppressing their cell viability and colony formation capacity. TF3 inhibited the tumorsphere formation capacity of A2780/CP70 and OVCAR3 CSCs in serum-free and non-adherent conditions. TF3 inhibited A2780/CP70 and OVCAR3 CSCs isolated from tumorspheres by decreasing their cell viability and upregulating the protein expression of caspase-3 and -7 in the cells. We also revealed that TF3 inhibited ovarian CSCs through Wnt/beta-catenin signaling pathway. Our results suggested that TF3 could inhibit ovarian CSCs and might be a potential agent for eradicating ovarian cancer.