Objective To establish a method for determination of ebracteolatain A content in the root of traditional Chinese medicine white Langdu, including Euphorbia fischeriana Steud and Euphorbia ebracteolata Hayata. Methods An HPLC-DAD method was created for determination at the following condition:the column was Agilent Zorbax SB-C₁₈ (4.6 mm×150 mm, 5 μm);mobile phase was acetonitrile:0.1% formic acid in water=55:45(V:V), isocratic elution, the flow rate was 1.0 mL·min^-1, the temperature of column was 25℃, the detection wavelength was set at 290 nm, the injection volume was 20 μL, and the running time was 20 min. Results Ebracteolatain A was separated from the interference in the baseline, and the linear range was 4.545-227.3 μg·mL^-1, with the linear correlation being 0.9999 for ebracteolatain A. The result of intra-day and inter-day precisions were both within 2%(n=3), and the average recovery was (99.14±3.4)%(n=6). The content of ebracteolatain A in two batches of Euphorbia fischeriana Steud and Euphorbia ebracteolata Hayata were (56.73±1.09) μg/g, (18.98±2.11) μg/g and (235.2±2.4) μg/g (n=6), respectively. Conclusion The present method is simple, rapid, accurate and convenient for determination of ebracteolatain A in the root of traditional Chinese medicine white Langdu.