Raman reporter‐based surface‐enhanced Raman scattering (SERS) has been used to investigate the detection of one member of a class of the cationic surfactant quaternary ammonium compounds, alkyl dimethyl benzyl ammonium chloride, also known as benzalkonium chloride (BAC). For this study, SERS was performed using silver nanoparticles (AgNPs). Density functional theory (DFT) of BAC on Ag6 clusters was introduced to estimate binding at the level of the B3LYP/LANL2DZ basis sets. As the chain length increased from n = 8 to 18, the binding energies were expected to increase by 2.1 kcal/mol. Methylene blue (MB) Raman reporter molecules were subsequently treated on BAC thin‐film‐covered glass surfaces. Atomic force microscopy (AFM) images were used to determine the coverage of BAC on the glass, and field‐emission electron microscope (FE‐SEM) images revealed the surface morphologies of the SERS substrates. A standard calibration curve was prepared to quantify BAC within a concentration range of 0.0036805 to 36 805 ppm (r2 ≥ 0.99), according to the BAC‐correlated SERS intensities of MB Raman peaks at ~1620 cm−1 adsorbed on AgNPs. A molecular dynamics (MD) simulation supported strong adsorption of BAC on glass surfaces.