Aim To establish a simplified extraction method from biological sample, and quantify sphingosine 1-phosphate (S1P) at the level of nanogram.Methods The methanol precipitation combing with ultrasonic extraction method was used to extract and separate S1P, then the supernatant was analyzed by liquid chromatography mass spectrometry (LC-MS) under the selective ion monitoring mode.Results The extraction method was effective and time saving, the limitation of quantification of S1P was 159.7 pg The S1P content in human plasma was 128.8 ± 33.3 μg/L, and the S1P content in plasma, heart, liver, lung, kidney and brain of the C57 BL/6 mouse was 181.8 ± 21.1 μg/L, 37.6 ± 2.8 ng/g, 54.9 ± 4.9 ng/g, 230.1 ± 60.5 ng/g, 21.5 ± 6.7 ng/g and 102.2 ± 23.8 ng/g, respectively.Conclusion Methanol precipitation combing with ultrasonic extraction could effectively separate S1P from plasma and organ, and the extract could be quantified by LC-MS directly. This method is effective with good reproducibility, and is suitable for quick quantitative analysis of a great deal of biological sample.