© 2023, Institute of Chemistry, Slovak Academy of Sciences.The combination of nanotechnology and sensor can allow the easy detection of diagnostic biomarkers, such as enzymes and proteins even the low abundance in biological fluids. The using a surface plasmon resonance (SPR) sensor system and graphene oxide (GO)-based molecular imprinting technology show a label-free, real-time, excellent sensitive, selective detection of the target molecule. The SPR sensor system occurs of a 50 nm Au nanofilm coated with a thin layer film of GO-based Lysozyme-imprinted composite nanofilm. The use of the GO-based SPR sensor for the real-time and selective determination of proteins was shown by utilizing Lysozyme as a model protein. Lysozyme is found in biological fluids including serum, saliva, tears, and urine which is called the “body's own antibiotic”. Lysozyme is an enzyme found in biological fluids, which in renal diseases as well as in a number of inflammatory gastrointestinal diseases, upregulated in leukemia. Therefore, the detection of Lysozyme in low concentrations plays an important role. The SPR sensor has high specificity and low-cost technology. In this work, a GO-based SPR sensor is designed for sensitive detection of Lysozyme using N-methacryloyl-(l)-phenylalanine as a hydrophobic composite's recognition element. The characterization of the SPR chip was obtained by Attenuated Total Reflection-Fourier Transform Infrared, Transmission electron microscopy, Ellipsometry and water contact angle measurements. Then, the aqueous Lysozyme solutions were studied. Different concentrations of Lysozyme solution were utilized to calculate kinetically. In this study, the sensor showed unique selectivity and recognition for the Lysozyme. The limit of detection value was reported 0.015 nM.