Folic acid (FA) is an ingredient that must be added to infant milk powder to avoid potential defects. Rapid, sensitive and reliable detection methods are needed to determined FA addition levels. Thus, this study established a microsphere immunochromatographic test strip for time-resolved luminescence detection (TRLM-ICTS) based on carboxyl-functionalized time-resolved luminescent microspheres (Eu-TRLMs) prepared by a one-step method as fluorescent markers for the immediate quantitative detection of FA in milk powder. Eu-TRLMs prepared by the one-step method showed good dispersion, high stability and strong fluorescence intensity, which is improving the sensitivity of TRLM-ICTS. In the performance evaluation of TRLM-ICTS, the detection limit was 0.487 ng mL−1, the recovery rate was 97.3–105 %, and the actual sample detection results were in line with those of UPLC-MS/MS. TRLM-ICTS has the advantages of rapid, high sensitivity and strong specificity and could as a practical quantitative detection method for the detection of FA in milk powder.