As a synthetic broad-spectrum antibiotic, chloramphenicol (CAP) is widely used in the prevention and treatment of bacterial diseases in aquaculture and animal husbandry, which might lead to severe water contamination and thus threaten our health. Herein, a novel electrochemical strategy for CAP detection is proposed with a sensor constructed based on the anodic peak (about -0.56 V) by modifying silver nanoparticles (AgNPs) and thiophene (TP) on a glassy carbon electrode (GCE) as a synergistic amplification unit with a simple step-by-step electrodeposition technique. Electrochemical methods, scanning electron microscopy, and X-ray energy dispersive spectroscopy were applied to characterize the as-prepared sensor. The TP/AgNPs/GCE sensor was used for CAP detection by DPV in the concentration range of 100.0 to 1600.0 µM, the limit of detection was 33.0 µM, and the sensitivity was 0.290 μA·µM-1·cm-2. In addition, the sensor has the advantages of simple preparation, low cost, good repeatability, stability, and anti-interference. It has been used for the detection of CAP in lake water with a recovery of 101.80 to- 104.85%, and the relative standard deviation was lower than 1.22%, which confirms that the sensor has good practicability.