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In-liquid biosensing is the new frontier of cells real time monitoring and biomarkers detection. In order to improve the stability and electrical properties of an Electrolyte Gated Organic Field Effect Transistor (EGOFET) biosensor, in this study we investigate the effect of the solvent and of the substrate modification on thin films of organic semiconductor Poly(3-hexylthiophene) (P3HT). The studied surface is the relevant interface between the P3HT and the electrolyte acting as gate dielectric for in-liquid detection of an analyte. AFM and XPS characterizations were employed to study the effect of two solvents (toluene and 1,2-dichlorobenzene) and of the adhesion promoter (Ti prime) on the morphological structure and electronic properties of P3HT film. Combining the results from the surface characterizations with electrical measurements, we investigate the changes on the EGOFET performances and stability in DI water with an Ag/AgCl gate electrode.