Low-dimensional films of graphene oxide (GO)-prussian blue (PB) hybrids were deposited on graphite screen-printed electrodes (SPEs) using a true bottom-up layer-by-layer approach that combines the self-assembly with the Langmuir-Schaefer method. A water dispersion of GO was used as subphase in a Langmuir-Blodgett (LB) deposition system while octadecylamine (ODA), that binds covalently with the GO, was injected at the air-water interface for the formation of an ODA-GO hybrid film onto graphite SPEs through hydrophobic interactions between the latter and the hydrophobic chain of ODA. Then, the outer side of the GO platelets were modified with a second ODA layer (which serves as an anchoring layer to PB through van der Waals attractive forces), immersed in a NaBH4 solution, and finally, the ODA-rGO-ODA layer was brought in contact with an aqueous PB solution. The as-fabricated ODA-rGO-ODA-PB nanostructured sensors were highly reproducible and showed a remarkable electrocatalysis towards reduction of hydrogen peroxide. Inherent limitations as regards the poor working stability of PB-modified sensors were effectively addressed. After an electroless ion-exchanged process in an aqueous solution of 0.1 mol L-1 RbCl (or CsCk) for 10 min, the resulting Rb(Cs)-modified assemblies showed an increased sensitivity and working stability, while the working stability was further improved at dimethyldioctadecylammonium-protected assemblies. The developed sensors showed a linear response over the concentration range 5-1000 mu mol L-1 H2O2 and were successfully applied at the amperometric determination of H2O2 in a mouthwash solution. The detection limit (S/N=3) and the relative standard deviation of the method were 2 mu mol L-1 H2O2 and <5% (n=10, 10 mu mol L-1 H2O2), respectively. The adopted fabrication approach combines low cost substrates, functional electrocatalysts and fully automated deposition techniques for producing, on a large scale, sensors offered a wide range of applications in chemical and biosensing in combination with suitable enzyme(s). (C) 2014 Elsevier Ltd. All rights reserved.