Objective To establish a convenient, reliable and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for detecting the content of 3 kinds of quorum sensing molecules (farnesol, tyrosol, and 3-indoleethanol) secreted by Candida albicans. Methods With carbamazepine as the internal standard, the samples were extracted with ethyl acetate and separated by gradient elution on Agilent Poroshell 120 EC-C₁₈ column (3.0 mm×100 mm, 2.7 μm) using Agilent 6470 triple quadrupole tandem mass spectrometry in positive ion monitoring mode. The mobile phase A was 0.1% formic acid in water, and the mobile phase B was acetonitrile. The elution gradient was 0-3 min 20%-40% B, 3-4 min 40%-80% B, and 4-8 min 80% B; flow rate was 0.5 mL/min; injection volume was 5 µL; column temperature was 25 ℃; the analysis period of each sample was 8 min; and the equilibrium time was 2 min. The mass spectrometry was performed using electrospray ionization source and positive ion multiple reaction monitoring mode. Results Farnesol, tyrosol and 3-indoleethanol had good specificity and linear relationship (all r＞0.999), and had good precisions and accuracy (intra- and inter-day precisions were ＜5% and absolute values of accuracy were ＜10%). The average recovery rate was 90%-115%. The stability under 3 conditions (4 h at room temperature, 24 h at 4 ℃ with autosampler, and 3 freeze-thaw cycles) met the requirements of the methodology. In the planktonic samples, the contents of the 3 kinds of quorum sensing molecules were increased with the growth of the strains. However, in the biofilm samples, the contents of farnesol and tyrosol were decreased at the mature stage of the biofilm. Conclusion This UHPLC-MS/MS method can be used to determine the contents of farnesol, tyrosol and 3-indoleethanol secreted by Candida albicans, and it provides a reference for rapid and accurate detection of quorum sensing molecules in fungi.