Recent advancements in MoS2 nanofilms have aided in the development for important water-related environmental applications. However, a MoS2 nanofilm-coated sensor has yet to have been applied for heavy metal detection in water-related environmental samples. In this study, a novel vertically aligned two-dimensional (2D) MoS2 (edge exposed) nanofilm was applied for in situ lead ion (Pb2+) detection. The developed sensor showed an excellent linear relationship toward Pb2+ between 0 and 20 ppb at -0.45 V vs Ag/AgCl using square wave anodic stripping voltammetry (SWASV) with the improved limit of detection (LOD) of 0.3 ppb in a tap water environment. The vertically aligned 2D MoS2 sensor exhibited improved detection sensitivity (2.8 folds greater than a previous metallic [Bi] composite electrode) with lower relative standard deviation for repetitive measurements (n = 11), indicating enhanced reproducibility for Pb2+ detection. The vertically aligned 2D MoS2 layers exhibited 2.6 times higher sensitivity than horizontally aligned 2D MoS2 (basal plane exposed). Density functional theory calculations demonstrated that adsorption energy of Pb on the MoS2 side edge was much higher (4.11 eV) than those on the basal plane (0.36 and 0.07 eV). In addition, the band gap center of vertical MoS2 was found to be higher than the Pb2+ → Pb reduction potential level and capable of reducing Pb2+. Overall, the newly developed vertically aligned 2D MoS2 sensor showed excellent performance for detecting Pb2+ in a real drinking water environment with good reliability.