The application of Langmuir–Blodgett (LB)-fabricated nanostructures on Ion selective Field Effect Transistors (ISFET) is of great interest since it permits the combination of a micro-scale device with a nanoscale material. In this article, the optimization of a LB-monolayer for calcium and sodium detection is carried out by characterization with XPS and AFM techniques, followed by electrochemical characterisation onto ISFET sensors. Physico-chemical modifications of the surface and good cation complexation of the ionophores fixed in the monolayers were observed with XPS studies. AFM images showed the presence of a unhomogeneous layer all over the ISFET surface. The electrochemical response of those monolayers onto ISFETs demonstrated the feasibility of this technology for sensor membrane fabrication and it opens further studies for other kind of devices such as optochemical sensors.