The rapid synthesis of gold nanocrystal with high catalytic activity is still a great challenge to improve the analytical performance of electrochemical sensors. In this paper, gold-arginine-threonine functionalized graphene quantum dot hybrid (Au-Arg-Thr-GQD) was synthesized by a simple and fast one-step method using Arg-Thr-GQD as reductant, stabilizer and crosslinker. The Au-Arg-Thr-GQD offers mulberry-shaped structure by the interconnection and self-assembly of Arg-Thr-GQD and small-sized Au nanocrystals. The unique structure significantly improves the catalytic activity of hybrid. Au-Arg-Thr-GQD was used as a sensing material for electrochemical analysis of imidacloprid. Under the optimal conditions, the differential pulse voltammetry peak current linearly increased with the increasing of imidacloprid concentration in the range of 0.1–20 μM with the detection limit of 0.011 μM. The proposed imidacloprid sensor shows high selectivity, good stability and reproducibility, and has been successfully applied to the determination of imidacloprid in cabbage and cucumber samples.