Rapid and on-site detection of chloramphenicol (CAP) is of great significance for food safety. Herein, we established an affordable and portable homogeneous fiber optic chemiluminescent immunosensor (hoFOCI) by streamlining the immunoassay and integrating the detection platform. By immobilizing the artificial antigens of CAP and the luminescent functional groups onto the fiber optic, the biorecognition and signal conversion could be performed without any washing steps, which greatly reduced the time consumption and simplified the operation. For chemiluminescence signal detection, a low-cost and integrated detection platform was constructed, which was designed to be compatible with fiber optic probe. The hoFOCI had a good linear response to CAP in the concentration of 10–106 pg/mL, with a detection limit of 2.95 pg/mL. The feasibility was also validated by assaying CAP in real honey samples, and a high accuracy and reproducibility were obtained. Therefore, the proposed hoFOCI is expected to be appropriate for rapid and on-site detection of CAP in food stuffs. Display omitted •A novel hoFOCI for rapid and on-site detection of chloramphenicol was developed.•Homogeneous assay offered a simple analysis procedure without washing steps.•Integrated hoFOCI device was low-cost and field operable.•High sensitivity, accuracy and stability were obtained for chloramphenicol analysis.