UQCRFS1 has been reported to be highly expressed in gastric and breast cancer, but the mechanism remains unclear. The prognosis and biological functions of UQCRFS1 in ovarian cancer (OC) have not been evaluated. The expression of UQCRFS1 in EOC was detected by GEPIA and HPA websites, and the prognosis value was investigated by Kaplan–Meier analysis. Then the correlation between the UQCRFS1 gene and tumor-related signature were analyzed by Spearman correlation analysis and rank sum test. Subsequently, the expression of the UQCRFS1 gene in four ovarian cancer cell lines was detected. A2780 and OVCAR8 with the highest expression of UQCRFS1 were selected in the following biological experiments. Cell proliferation was detected by CCK8 assay, cell cycle and apoptosis were determined by flow cytometry, reactive oxygen species (ROS) production was detected by DCFH-DA, DNA damage gene mRNA expression was analyzed by RT-PCR, and AKT/mTOR pathway protein expression were also examined by western blot after siRNA transfection. We found that UQCRFS1 was high-expression in EOC and associated with poor prognosis. Spearman correlation analysis revealed that the high expression of UQCRFS1 is associated with the cell cycle, apoptosis, oxidative phosphorylation, and DNA damage. Further studies found that knockdown of UQCRFS1 cells reduced cell proliferation, cell cycle arrest at the G1 phase, increased proportion of apoptosis, ROS production, and expression of DNA damage genes, inhibited ATK/mTOR pathway. The study suggested that UQCRFS1 may be a candidated target for diagnosis and treatments in OC.