Penicillin G (PEN) is a widely used antibiotic for treating microbial infections. However, its extensive use in veterinary medicine can lead to accumulation in animal-derived products, particularly milk and meat. This highlights the urgent need for rapid and sensitive antibiotic detection methods. In this study, we employed DNA aptamers for the detection of PEN and for the analysis of aptamer specificity using a combined approach based on quartz crystal microbalance with dissipation monitoring (QCM-D) and localized surface plasmon resonance (LSPR). QCM-D measures changes in resonant frequency, Δf, and dissipation, ΔD, while LSPR monitors wavelength shifts in the extinction spectra corresponding to changes at the surface of gold nanoparticles (AuNPs). Thiolated aptamers were chemisorbed onto the surface of AuNPs with a diameter of 80 nm. In the presence of PEN, a redshift in the extinction spectra and a decrease in resonant frequency were observed, accompanied by an increase in dissipation due to surface viscosity effects. Significant changes in both acoustic and LSPR signals were observed at PEN concentrations as low as 1 nM. The limits of detection (LOD) for PEN, determined by QCM-D (3.0 nM, or 1.05 ng/mL)) and LSPR (3.1 nM, or 1.09 ng/mL), were similar and both were lower than the maximum residue limit (MRL) for PEN established by the EU (4 ng/mL).