A fluorescent single-domain antibody (fluobody), consisting of a single-chain variable fragment antibody (scFv) and a green fluorescent protein extracted from aequorea coerulescens (AcGFP), was produced and used to develop a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) for the detection of avermectins (AVMs). The scFv gene was prepared by cloning V_H and V_L genes from a hybridoma cell line (2C11) and then fused to the C-terminus of AcGFP (fluobody) with a flexible peptide linker (Gly₄Ser)₂ between the two domains. After expression and purification, the functional fluobody was used to develop a one-step FLISA protocol for the determination of AVMs in milk samples. The 50% inhibition concentration (IC₅₀) value and the limit of detection (LOD) of the optimized immunoassay for abamectin (ABM) were 2.13 and 1.07 ng mL⁻¹, respectively. Cross-reactivity studies showed that the fluobody exhibited high affinity for the other four AVMs. The recoveries from the spiked milk samples ranged between 86.8 and 125.0%, with relative standard deviation lower than 10.2%. Moreover, the developed FLISA was applied to field samples, followed by confirmation with liquid chromatography-fluorescence detection (LC-FLD) analysis. The consistency of results between the immunoassay and instrumental techniques in detecting the presence of AVMs near the detection limit of the FLISA indicated that the newly developed method is suitable for rapid screening of AVM contamination in food samples prior to chromatographic analysis.