Apramycin (APR) residue in food of animal origin can cause harmful effects on human health. In this study, a monoclonal antibody (mAb) was successfully produced using APR-BSA as immunogen, which was prepared by glutaraldehyde method. The mAb 2A2 showed low cross reactivity (<0.1%) with other aminoglycoside antibiotics, and its IC50 value was 0.35 ng/mL. Based on this mAb, a novel immunoassay in the format of immuno-affinity test column (IATC) was developed. The immune-affinity column filled with anti-APR antibody-Sepharose 4B gel was used as solid phase. APR in sample and HRP-APR conjugate compete with each other for the limited antibody on the column. The approach was able to give a naked-eye color signal from the detection of analyte. A blue color appears for negative results and no color for positive. The method was then successfully applied onto the detection of APR in animal-origin food. To further evaluate the assay, direct competitive ELISA (dcELISA) based on the same antibody was developed for comparison in different aspects. Compared to the dcELISA, the detection time of IATC is shorten to 20 min while the similar sensitivity for various samples was observed. The limit of detections (LOD) for raw milk, muscles and livers is 3 ng/mL, 3 µg/kg, and 10 µg/kg, respectively.