Abstract： Objective To synthesize 16α-[18F]fluoro-17β-estrogen(18F-FES) with high chemical and radiochemical purity using multifunctional radiolabeling module of Explora GN and separation module of Explora LC (Explora GN/LC dual module). Methods Explora GN/LC dual module and "one-pot" radiochemical process were applied for fully automated synthesis and separation of 18F-FES. The radiochemical purity of 18F-FES was analyzed by HPLC. The saturation binding test of 18F-FES with ER (ER-positive MCF-7 cell line) was applied to examine the biological activity of 18F-FES. Results 18F-FES synthesis procedure took 60 min, with radiochemical yield about 45% and radiochemical purity higher than 98%. The Kd and Bmax of MCF-7 cells with 18F-FES were (2.922±0.619) nmol/L and (4.193±0.360) nmol/L respectively. Conclusions 18F-FES can be automatically synthesized by Explora GN/LC dual module with high chemical purity and radiochemical purity. It would be a potential PET tracer to be used in estrogen-related molecular imaging.