The development of18F-fluorotetrazines, suitable for the radiolabeling of biologics such as proteins and antibodies by IEDDA ligation, represents a major challenge, especially for pre-targeting applications. The hydrophilicity of the tetrazine has clearly become a crucial parameter for the performance of in vivo chemistry. In this study, we present the design, the synthesis, the radiosynthesis, the physicochemical characterization, the in vitro and in vivo stability, as well as the pharmacokinetics and the biodistribution determined by PET imaging in healthy animals of an original hydrophilic18F-fluorosulfotetrazine. This tetrazine was prepared and radiolabelled with fluorine-18 according to a three-step procedure, starting from propargylic butanesultone as the precursor. The propargylic sultone was converted into the corresponding propargylic fluorosulfonate by a ring-opening reaction with18/19F-fluoride. Propargylic18/19F-fluorosulfonate was then subject to a CuACC reaction with an azidotetrazine, followed by oxidation. The overall automated radiosynthesis afforded the18F-fluorosulfotetrazine in 29–35% DCY, within 90–95 min. The experimental LogP and LogD7.4values of −1.27 ± 0.02 and −1.70 ± 0.02, respectively, confirmed the hydrophilicity of the18F-fluorosulfotetrazine. In vitro and in vivo studies displayed a total stability of the18F-fluorosulfotetrazine without any traces of metabolization, the absence of non-specific retention in all organs, and the appropriate pharmacokinetics for pre-targeting applications.