Self-assembled lipid structures are studied towards mimicking the cellular structure electronically. Applications of these platforms for different biosensing are rather limited. Here, we developed bilayer of N[1-(2,3-Dioleoyloxy)propyl]-N, N, N-trimethylammonium chloride (DOTAP) by non-covalent electrostatic interaction on the head groups of pure 3-mercapto propionic acid (MPA) and 6-mercapto hexanol (MCH) mixed monolayer on gold surface. The tethered layer was characterized by Atomic force microscopy (AFM), Transmission electron microscopy (TEM) and goniometry. Influence of gold nanoparticle on the ion channel activity of pore forming membrane proteins melittin (MTN) and valinomycin (VM) were evaluated by potential dependent impedance and voltammetric techniques. The gold nanoparticle (AuNP) helps to improve the ion transport property of membrane protein by blocking the monolayer defects and inserting membrane protein into the bilayer membranes. Label free DNA and protein sensing were made in addition to the ion channel activity. Protein concentration studies indicate the orientational requirement of the membrane protein for the effective ion transport. This behavior is useful as a diagnostic parameter in the label free protein toxins (MTN and VM) sensing in the presence of electroactive redox probe [Fe(CN)(6)](3-/4-) which showed the lowest detection limits of 1.5 pM respectively with high selectivity, sensitivity and reproducibility.