Chloramphenicol (CAP) as an amide-alcohol antibiotic is extensively used in aquaculture industries and can accumulate in the human body through the food chain and water sources, leading to various diseases. On-site inspection of CAP remains a challenge due to the lack of portable and sensitive sensing platforms. Herein, a bi-atomic matching catalyst that comprises atomic Fe uniformly distributed on an N-doped graphene matrix (A-Fe-NG) is synthesized. An electrochemical sensor based on A-Fe-NG is constructed for CAP, demonstrating a high sensitivity of 164.2 μA μM