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Biosensors based on Organic Field-Effect Transistors (OFETs) have attracted increasing attention due to the possibility of rapid, label-free, and inexpensive detection. Among all the different possibilities, inkjet-printed top-gate organic Field Effect Transistors-Based Biosensors (BioFETs) using a polymeric gate insulator have been seldom reported. In this work, a systematic investigation in terms of topographical and electrical characterization was carried out in order to find the optimal fabrication process for obtaining a reliable polymer insulator. Previous studies have demonstrated that the best electrical performance arises from the use of the perfluoropolymer Cytop™[12,13,14]. Consequently, a simple immobilization protocol was used to ensure the proper attachment of a model biomolecule onto the Cytop's hydrophobic surface whilst keeping its remarkable insulating properties with gate current in the range of dozens of pico-amperes. The top-gate inkjet-printed BioFETs presented in this study operate at threshold voltages in the range of 1–2 V and show durability even when exposed to oxygen plasma, wet amine functionalization treatments, and aqueous media. As a preliminary application, the inkjet-printed top-gate BioFETs is used for monitoring an immunoreaction by measuring changes in the drain current, paving the way for further use of this device in the immunosensing field. A systematic study and optimization of a simple functionalization procedure to attach antibodies onto Cytop film for obtaining inkjet printed top-gate BioFETs is presented. Image 1 • · The proposed inkjet-printed organic transistor-based flexible sensor delivers a label-free response for immunosensing applications. • It is shown that the use of the perfluoropolymer Cytop as gate insulator presents the best electrical performance in terms of leakage current. • A functionalization procedure is presented to attach biomolecules onto the Cytop surface while keeping its remarkable insulating properties. • The BioFET has been evaluated as a proof-of-concept sensor to detect a model biomolecule through drain current changes. [ABSTRACT FROM AUTHOR]