Dexamethasone (DEX) is widely used because of its anti-inflammatory, anti-endotoxin, anti-shock, and stress-enhancing response activities. It can increase the risk of diabetes and hypertension if it is abused or used improperly. However, there is a lack of sensitive and rapid screening methods for DEX in food. In this study, a time-resolved fluorescent microspheres immunochromatographic assay (TRFM-ICA) integrated with a portable fluorescence reader was developed for the quantitative detection of DEX in milk and pork. The cut-off values of the TRFM-ICA were 0.25 ng/mL and 0.7 µg/kg, respectively. The limits of quantitation (LOQs) were 0.003 ng/mL and 0.062 µg/kg, respectively. The recovery rates were 80.0–106.7%, and 78.6–83.6%, respectively, with the coefficients of variation ranging 6.3–12.5%, and 7.5–10.3%, respectively. A parallel experiment for 20 milk and 10 pork samples with LC-MS/MS was carried out to confirm the performance of the on-site application of the developed TRFM-ICA. The results of the two methods are basically the same; the correlation (R2) was >0.98. The establishment of TRFM-ICA will provide a new sensitive and efficient technical support for the rapid screening of DEX in food.
