Graphene particles were synthesized from biomass, i.e., wood powder, for the first time in this work for analyzing milk protein content in samples such as cow milk and pocket milk. The production of graphene on large scale is one of the biggest challenges. Graphene synthesis by a low-cost method is achieved by thermal treatment of wood powder. Label-free detection of milk by graphene using UV-Visible spectroscopy is reported. This method of detection of milk is rapid, highly sensitive, and more economical. The large specific surface area of graphene allows for very good adsorption of target biomolecules. Though graphene is reported widely for its remarkable properties such as high electrical and thermal conductivity, high strength, anti-corrosion, and sensitivity, its applications are less explored. Graphene is synthesized by heating wood powder to a high temperature of about 800 °C. The structural and morphological studies are performed using XRD, FTIR, and SEM analysis. The protein content in milk is measured by optical sensing using graphene as the sensing material. Raw cow milk and commercial pocket milk are selected, and their quality is analyzed using UV-Visible spectroscopy. The milk samples are detected using the surface plasmon resonance (SPR) peak of graphene through the non-enzymatic method. The analysis of the SPR peak at 282 nm clearly shows the ability of graphene as a bio-molecular recognition element. The intensity of the absorbance increases linearly when the amount of milk increases from 0.25 to 1 ml. Thus, graphene can replace noble metal nanoparticles in the field of biosensing.