The antibiotic penicillin G (PEN) is commonly used for the treatment of microbial diseases. However, its extensive application in veterinary medicine can cause infiltration into food, especially milk and meat. Therefore, there is an urgent need for rapid and sensitive methods for antibiotic detection. In this study, we used DNA aptamers specific to PEN for its detection using a combined approach based on an acoustic method, quartz crystal microbalance with dissipation (QCM-D), and an optical method, localized surface plasmon resonance (LSPR). QCM-D measures changes in the resonant frequency, Δf, and dissipation, ΔD, while LSPR monitors the shifts in the wavelength 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. Upon the presence of PEN, a shift to a longer wavelength and a decrease in resonant frequency was observed, accompanied by an increase in dissipation due to surface viscosity effects. Significant changes in the acoustic and optical signals were observed down to a PEN concentration of 1 nM, which is lower than the maximum residue limit (MRL) for this antibiotic established by the EU (4 µg/kg, ~ 12 nM). The sensor selectively detects PEN, as demonstrated in experiments with a non-specific antibiotic, oxytetracycline.