In this research, we fabricated a sensitive monoclonal antibody (mAb) 2C3 that targeted etomidate (ET) and metomidate (MT) to establish a lateral-flow immunoassay (LFIA) that incorporated fluorescent microsphere sensors, enabling both the qualitative and quantitative detection of ET and MT within 10 min. Analysis indicated that the visual colorimetric values for ET and MT in water samples were 0.3 μg kg-1, respectively, with quantitative detection ranges of 0.08 to 1.31 μg kg-1 and 0.08 to 2.21 μg kg-1, respectively. The visual colorimetric values for ET and MT in urine samples were 0.3 and 1 μg kg-1, respectively, with quantitative detection ranges of 0.12 to 3.13 μg kg-1 and 0.09 to 2.98, respectively, while for ET and MT in serum samples were 3 μg kg-1, with quantitative detection ranges of 0.15 to 21.2 μg kg-1 and 0.08 to 13.8 μg kg-1, respectively. Recovery tests and detection were conducted in water, urine and serum samples, validating the reliability of this method in clinical samples, consistent with those obtained from LC-MS/MS. Collectively, our novel LFIA provides a promising option for rapid on-site detection of ET and MT.