Introduction: Silymarins are the main active ingredient in milk thistle extract, reflecting the quality of extract products. The detection methods for silymarins in different pharmacopoeia standards may vary, which may lead to inconsistent detection results. Method: Due to the lack of research on related fingerprint spectra, its qualitative work is hard to carry out. Hence, based on existing standard methods, an HPLC method was established by optimizing the gradient program of the mobile phase. Results: The effectiveness of this method and the fingerprint was also studied. The similarity of 31 batches of samples ranged from 0.791 to 1.000, indicating the characteristic spectrum formed can be well used for qualitative analysis. Conclusion: Under optimal analytical conditions, the six silymarin compounds in the samples can achieve good baseline separation from the matrix with good resolution. At the same time, the LOD was 25 mg/kg, and the recovery rates at three levels ranged from 92% to 108%, with RSDs less than 4%. The fingerprint spectrum and content determination method established in this study had good repeatability and high accuracy, which can provide a new method for quality control of milk thistle extract.