Pregnanediol-3-glucuronide (PdG), as the main metabolite of progesterone in urine, plays a significant role in the prediction of ovulation, threatened abortion, and menstrual cycle maintenance. To achieve a rapid and sensitive assay, we have designed a competitive model-based time-resolved fluorescence microsphere-lateral flow immunochromatography (TRFM-LFIA) strip. The optimized TRFM-LFIA strip exhibited a wonderful response to PdG over the range of 30-2,000 ng/mL, the corresponding limit of detection (LOD) was calculated as low as 8.39 ng/mL. More importantly, the TRFM-LFIA strip was innovatively used for the quantitative detection of PdG in urine sample, and excellent recovery results were also obtained, ranging from 97.39% to 112.64%. The TRFMLFIA strip possessed robust sensitivity and selectivity in the determination of PdG, indicating the great potential of being powerful tools in the biomedical and diagnosis region.