In this study, selective and sensitive Penicillin G (PEN-G) antibiotic detection from both aqueous solution and milk samples using molecular imprinting technique was performed by surface plasmon resonance sensor. For this purpose, PEN-G imprinted poly(2-Hydroxyethyl methacrylate-N-methacroyl-(L)-cysteine methyl ester-gold nanoparticles-N-methacryloyl-L-phenylalanine methyl ester (MIP-AuNPs) nanosensor was prepared. Control experiments were carried out via the nanosensor without the addition of AuNPs (MIP) to examine the effect of gold nanoparticles (AuNPs) incorporated to increase the surface plasmon resonance signal response. Moreover, to evaluate the imprinting efficiency, non-imprinted (NIP-AuNPs) nanosensor was designed using the same polymerization recipe except the addition of the PEN-G molecule. Characterization studies of MIP, MIP-AuNPs and NIP-AuNPs nanosensors were performed with FTIR-ATR spectrophotometer. Also, MIP-AuNPs and NIP-AuNPs nanosensors were characterized by atomic force microscopy, ellipsometer and contact angle measurements. Imprinting efficiency (I.F: 7.83) for the MIP-AuNPs nanosensor was determined by comparing it with the NIP-AuNPs nanosensor. The MIP-AuNPs nanosensor was 9.87 times more selective for the target PEN-G molecule than amoxicillin, and 16.78-times than ampicillin. In addition, the amount of PEN-G in milk selected as a real sample was measured by spiking 5 ppb PEN-G into the milk.