Endocrine-disrupting chemicals (EDCs) in the secondary effluent discharged from wastewater treatment plants are of great concern when water reuse is intended. The combined process of ozone (O3) and ultrafiltration (UF) is a promising EDC removal method. The removal efficiency of five EDCs using O3, UF and their combination were investigated and compared. The five EDCs were estrone, 17β-estradiol, estriol, 17α-ethynyl estradiol and bisphenol A, which are typically present in secondary effluent. Results showed that organic matters in secondary effluent became easier to be removed by the combined process, with ultraviolet absorbance reduction enhanced by 11%–18% or 24%–26% compared to the UF or O3 alone. The removal efficiency of EDC concentration, estrogenicity and acute ecotoxicity by the combined process was 17%–29% or 54%–92%, 19% or 73%, 40% or 60% greater, respectively, than that of the O3 or UF alone. Particularly, when EDCs were treated by the combination of O3 and UF, about 100% EDC removal efficiency was achieved. Overall, the combined application of O3 and UF offers an effective approach to control the concentration and toxicity of EDCs in secondary effluent.