Abstract: To explore novel synthetic routes of 16-epiestriol (Estra-1, 3, 5(10)-triene-3, 16β, 17β-triol), we used the estrone as the starting material. After some simple synthetic steps, 16-epiestriol was yielded. Identification and purity of intermediates in the synthetic routes were characterized by melting point and ¹H NMR spectrum analysis, respectively. In addition, the structure of 16-epiestriol was modified and radiolabeled with ¹⁸F-fluoride to yield ¹⁸F-FES. The corresponding quality control analysis of the injection were performed. The yield of two synthetic routes of 16-epiestriol were about 20% starting from estrone. Radiosynthesis of ¹⁸F-FES was finished in 60 minutes with a radiochemical yield of (30±4)% and radiochemical purity greater than 99%. The ¹⁸F-FES injection was colorless and clear and the pH value was 6.5-7.5. The specific activity of the injection saline was (1.75±0.25) Ci/μmol. In this study, 16-Epiestriol was yielded with a relatively high yield in two novel routes. ¹⁸F-FES was finally yielded after structure modification of 16-epiestriol and the corresponding radiolabeling procudure. The quality control results of the ¹⁸F-FES injection could meet the need in clinical examination.