Nanotechnology has attracted immense interest in developing nanomaterial-based biosensors. This surge is attributed to the characteristic features of nanomaterials which involve its size and large surface to volume ratio. Nanomaterials like nanoparticles, carbon nanotubes and quantum dots are used widely in developing the biosensing system particularly for the analysis of food samples. The chemical synthesis of nanoparticles has certain limitations, thus prompting us to adopt green synthesis which offers nontoxic and eco-friendly procedures. In the present study, gold nanoparticles were synthesised using Syzygium aromaticum extract in which the main constituent eugenol acts as a reducing agent. Gold nanoparticles were characterised by UV–vis spectroscopy, transmission electron microscopy, dynamic light scattering and Fourier transformed infrared spectroscopy. They exhibited polygonal and triangular shapes with size range from 4 to 150 nm. Further, they were functionalised with a 3–5-nm thick layer of cysteine to increase the stability of enzyme for the development of biosensor. In our investigation, we observed that gold nanoparticles were able to enhance the response of the urea biosensor by up to 60 % owing to increased surface area of matrix and improved stability of the enzyme. To ensure milk safety, we developed a colorimetric biosensor for determining urea in milk. The developed biosensor is a simple, disposable and economical detection system, the response of which is more distinctive at lower urea concentrations with a reliability of 94.6 %.