Determination of beta-lactoglobulin in milk products is very important because beta-lactoglobulin is the main ingredient in whey-based protein powders. However, beta-lactoglobulin is a dangerous food allergen. On such an occasion, the determination of beta-lactoglobulin is coming into prominence and electrochemistry is a good alternative for this purpose because of its simple, economic and rapid response. In this work, a graphene oxide modified pencil graphite electrode is developed to determine beta-lactoglobulin based on the current signal of known concentration of hydrogen peroxide. Cyclic voltammetry technique is performed to obtain the electrochemical behavior of beta-lactoglobulin. Linear range, limit of detection and limit of quantification were calculated according to the calibration curve of various amounts of beta-lactoglobulin. Ultraviolet-visible spectroscopy technique is also used to investigate the absorption behavior of beta-lactoglobulin in various biological macromolecules including whey proteins. The proposed graphene oxide modified pencil graphite surface is successfully applied to determine beta-lactoglobulin in real milk sample, so a new methodology based on a newly developed electrochemical technique is described as a promising alternative in diary products.