Disclosed is a method of extracting all metabolites existing in a biological sample to detect a comprehensive difference between metabolites of a control group and a test group, thereby verifying significance thereof. More particularly, the invention comprises a first step of analyzing a fraction extracted with a solid phase extraction method, a fraction obtained by extracting the remnant fraction with a liquid-liquid extraction method and fractions extracted with the liquid-liquid extraction method at two pH different from each other after the hydrolysis, with a gas chromatography-mass spectrometry; a second step of converting the chromatogram result into numerical values capable of being statistically processed; and a third step of analyzing the numerical values with a principal component analysis (PCA) and a discriminant analysis (DA) to detect a difference between the control group and the test group. According to the invention, the difference between the two groups can be comprehensively detected with the areas of the peaks on the chromatogram, without a standard material or verified quantitative method. Therefore, it is possible to detect the metabolic change in the organism due to the disease or gene mutation, without an accurate quantitative analysis of the metabolites.