The invention relates to the technical field of dairy product detection, in particular to a method for simultaneously detecting three
steroid hormone substances in
raw milk. The three
steroid hormone substances are estradiol, ethinyl estradiol and
estrone respectively, and the detection method comprises the following steps: 1) removing
protein in
raw milk to obtain a sample solution to be detected; (2) carrying out qualitative and quantitative analysis on the three
steroid hormone substances in the sample solution to be detected by adopting a
high performance liquid chromatography-
ultraviolet detection method; wherein the
qualitative analysis is based on the
retention time of each substance, and the quantitative analysis is based on a standard curvilinear equation to calculate the concentration. According to the detection method, the dependence on expensive instruments and professional operators is remarkably reduced while high sensitivity and accuracy are guaranteed, the cost is low, and the method is particularly suitable for rapid screening of grassroots units and large-batch samples.