The use of graphene has leapt forward the materials field and the functional modification of graphene has not stopped. In this work, β-lactoglobulin (BLG) was used to functionalize reduced graphene oxide (RGO) based on its amphiphilic properties. Also, trimetallic PtAuPd nanoparticles were reduced to the surface of BLG-functionalized RGO and formed BLG-PtAuPd-RGO nanocomposite using facile synthesis. Transmission electron microscopy, energy-dispersive X-ray spectroscopy and Fourier transform infrared spectra were used to characterize the nanocomposite. Electrocatalytic analysis was evaluated through cyclic voltammetry and chronoamperometry methods. We developed a glucose sensor by fabricating GOD-BLG-PtAuPd-RGO/glassy carbon (GC) electrode. It presented a remarkable sensitivity of 63.29 μA mM−1 cm−2 (4.43 μA mM−1), a wider linear range from 0.005 to 9 mM and a lower detection limit of 0.13 μM (S/N = 3). Additionally, the glucose sensor exhibited excellent testing capability in human serum samples.