Chloramphenicol (CAP) is a synthetic antibiotic that inhibits protein synthesis by binding to the ribosomes of bacteria to achieve the purpose of antibacterial. Long-term intake of residual CAP animal-derived food can lead to anemia and leukemia in the human body and can also cause the body to develop drug resistance, which will seriously endanger human health. Many countries have regulations prohibiting the detection of CAP in livestock products. Therefore, designing a more rapid, simple and highly specific CAP detection method is of great significance. In this paper, the thiolated aptamer of CAP (SH-Apt) was used to modify the silver nanorod array chip(chip)as the SERS substrate, and the DNA hybridization indicator methylene blue (MB) as the Raman reporter, A novel high-specificity CAP-SERS aptamer sensor was constructed by utilizing the competitive binding relationship between CAP, CAP aptamer complementary strand (cDNA) and CAP aptamer (Apt). The chip and CAP-SERS aptamer sensor were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and EDS spectroscopy. The results showed that a large amount of silver elements were uniformly distributed on the surface of the chip and the CAP-SERS aptamer sensor was successfully prepared. Detection of CAP standards at room temperature, the results of the sensor-related performance analysis suggest that, with an increase of CAP concentration (0.001～10 ng·mL-1) caused the decreased SERS signal at 1 624 cm-1(ISERS) intensity (ISERS=-971logc+1 983). A good negative correlation (R2=0.991) was achieved, and the limit of detection (LOD) was as low as 0.2 pg·mL-1 (S/N=3), the Raman enhancement factor EF=1.01×107. Further indicating that the substrate has good Raman enhancement. The sensor is used to detect individual CAP tablets and CAP in human and pig sera, spik experiments were performed, and the results were satisfactory. The recovery and relative standard deviations(RSD) were 91.2%～120.5% and 0.97%～8.1%, which proved that the sensor had good accuracy. The sensor is intended to be used for the rapid quantitative detection of CAP in real samples because of its simple manufacture, high sensitivity, strong selectivity, good reproducibility, good stability, and fast detection speed. It provides a new idea for detecting CAP.