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Sulfamethazine (SMZ), widely used for disease prevention of bees, is harmful to consumers when excess SMZ residues are present in honey. In this study, a novel competitive lateral flow immunoassay (LFIA) was established by using aggregation-induced emission fluorescent microsphere (AIEFM) as label for the detection of SMZ. Results indicated that the limit of detection (LOD) for the developed competitive AIEFM-LFIA in honey was 0.028 ng/mL, and the corresponding linear range was 0.05−5 ng/mL. Recovery experiments showed that the average recovery range was 75.6–95.1% with coefficient variations of 5.7–12.9% in honey. The developed competitive AIEFM-LFIA was compatible with HPLC-MS/MS in detecting SMZ in 15 real samples from honey. In general, the proposed AIEFM-LFIA is well suited for the competitive format and has great potential for the detection of other small targets with single antigenic site in practical application.