Antibiotic overuse poses a serious food safety problem. Therefore, it is of great importance to develop efficient assays that respond to antibiotics to establish early-warning mechanisms. Here, we prepared a three-dimensional (3D) porous reduced graphene oxide (pRGO) modified electrode, which was characterized by scanning electron microscopy and transmission electron microscopy. As a result of the introduction of the 3D pRGO film, the electrocatalytic activity was considerably improved, which could efficiently trigger the redox reaction of rifampicin (RIF). By employing differential pulse voltammetry, the reduction peak current of RIF showed a good linear relationship with the logarithm of the RIF concentration in the range 1.0 × 10-9 to 1.0 × 10-7 mol L-1. The linear equation was ip (-10-6 A) = 3.11 + 0.28 log cRIF (R2 = 0.9908) with a detection limit of 2.7 × 10-10 mol L-1 (S/N = 3). Additionally, the final electrode displayed long stability, good reproducibility and high selectivity, and could detect trace RIF in milk with satisfactory results. This study reveals the great potential in utilizing 3D pRGO to develop efficient electrochemical sensors for safeguarding food safety.