Food contamination by veterinary drug residues is a world-wide public health concern and requires continuous monitoring. In this study, we developed a biotin–streptavidin-amplified ELISA (BA-ELISA) using a produced monoclonal antibody for detecting pirlimycin residues in beef muscle, milk and honey. The IC50 value of the BA-ELISA was 1.6 ng/mL for pirlimycin in buffer, and the sensitivity was improved 3 times compared to traditional ELISAs. The optimized BA-ELISA can be used to quantitate trace amounts of pirlimycin residues in beef muscle, milk and honey. This method had a limit of detection (LOD) of 4.45 μg/kg in beef muscle, 1.65 μg/L in milk and 2.75 μg/kg in honey. The average recovery of the BA-ELISA ranged from 78% to 97%, and the coefficient of variation ranged from 5.3% to 13.5%. The developed BA-ELISA method was validated using LC–MS/MS, and the BA-ELISA can be used for routine screening analysis of pirlimycin residues.