The present invention provides an enzyme-linked immunoassay kit for chloramphenicol detection. The enzyme-linked immunoassay kit comprises an enzyme label plate coated with coating antigen, an enzyme marker, a specific chloramphenicol antibody working solution (when the enzyme label plate is coated with antigen and the enzyme marker is enzyme-labeled anti-antibody or the enzyme label plate is coated with anti-antibody and the enzyme marker is enzyme-labeled antigen, the kit contains the specific chloramphenicol antibody working solution), chloramphenicol standard solutions, a substrate coloration solution, a termination solution, a concentration washing solution and a concentration reconstituted solution. The present invention further discloses a method for detecting chloramphenicol by using the enzyme-linked immunoassay kit. The method comprises: carrying out a sample pretreatment, adopting the kit to detect, and finally analyzing a detection result. With the enzyme-linked immunoassay kit, chloramphenicol residue in animal tissues (muscles, livers and the like), delicatessen, aquatic products, royal jelly, urine, feeds, milk and other samples can be detected, characteristics of simple operation, low cost and high sensitivity are provided, and the kit and the method can be applicable for screening and on-site monitoring of a large number of samples.