In this exploratory study, Langmuir-Blodgett (LB) films of gold nanoparticles (Au NPs) were utilized for the first time to detect botulinum neurotoxin (BoNT) based on localized surface plasmon resonance (LSPR), acting as biosensors. Monolayers of Au NPs were initially transferred onto a transparent polymer substrate using the LB technique. This substrate was then used as the base material for subsequent depositions of capping ligands, and eventually, the BoNT at different concentrations. Upon each deposition, LSPR signals were recorded employing UV-Vis spectroscopy. As a result, it was demonstrated that the LB films transferred at a surface pressure of 35 mN m-1 were the optimal choice, capable of detecting BoNT at a concentration as low as 1 pg ml-1. Furthermore, it was discovered that the formation of Au NP clusters reduced the sensing capacity of the LB films. This sensor offers advantages such as easy fabrication and a quick detection process that utilizes visible light.