The invention relates to the technical field of chemical detection, and provides a method for detecting the content of conjugated estrogens in pregnant mare urine. Carbonate in the pregnant mare urinecan form neutral salt, water and carbon dioxide gas when encountering inorganic acid; the column efficiency is improved, so that the problems that 30 samples cannot be measured each time, the columnpressure is increased, the column efficiency is reduced and the like when the content of conjugated estrogens in pregnant mare urine is measured by HPLC can be solved, the measurement accuracy of thesample content is ensured, and reliable data is provided for production. The pH value is adjusted to 7.0-8.0 by using an alkaline substance because: a, the existence state of bound estrogen is relatively stable under an alkaline condition; b, the pH value of the pregnant mare urine is about 7.5-8.5, and the pH value of the pregnant mare urine is adjusted to be slightly alkaline (7.0-8.0), so thatthe pregnant mare urine is closer to the original conditions of the pregnant mare urine; and c, the authenticity and the reproducibility of a detection result have advantages when the sample is slightly alkaline.