Objective To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of seven steroid hormones in sera of patients with primary hepatocellular carcinoma. Methods The serum samples were extracted with ethyl acetate, and then derivatized with dansyl chloride. Chromatographic column was Agilent Poroshell 120 EC-C₁₈ (2.1 mm×150 mm, 2.7 μm) column, mobile phase was acetonitrile and 0.1% formic acid-water solution, flow rate was 0.3 mL/min, the column temperature was 35℃, and the MS detection was selected in dynamic MRM mode. Results Seven steroid hormones (estradiol, estriol, estrone, hydrocortisone, testosterone, androstenedione and progesterone) were baseline separated within 8 min, which possessed good linear relationship (r>0.99) within the linear concentration range; the intra-day and inter-day precisions were less than 15%, the recoveries were ranged from 80% to 119%, the matrix effect was 85%-112%. The detection results were stable when the samples were examined 6 h after collection, 24 h after treatment at room temperature, after three freeze-thaw cycles, and 30 days when stored at -80℃. Conclusion The current LC-MS/MS method is simple, accurate, sensitive and reproducible, and it can be applied to determine steroid hormones in the sera of patients with primary hepatocellular carcinoma.