Human milk oligosaccharides (HMOs) have become a hot topic in the field of maternal and infant nutrition and health research, and are also widely used as innovative functional ingredients in infant formulas, but their complex structure makes its isolation and analysis difficult. This paper presents a simple method for the extraction and analysis of HMOs. The effect of the presence of lactose on the determination of HMO content was investigated by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Human milk is dynamically enriched with oligosaccharides using graphite carbon pipes to reduce the interference of lactose with the assay. It was found that the mass concentrations of HMOs obtained after removing more than 90% of lactose were more accurate. 2’-Fucosyllactose (2’-FL) and 3-fucosyllactose (3-FL) were linear in the range of 1.50–100.00 μg/mL (R2 > 0.997), and the remaining eight HMOs were linear in the range of 0.75–50.00 μg/mL (R2 > 0.995). Precision (relative standard deviation (RSD)) values for each HMO detection result ranged from 1.47% to 5.26%. Accuracy is from 98.74% to 102.26%. This analytical technique is a sensitive, precise, and accurate quantitative method for the quantification of ten HMOs in human milk. This method can provide accurate and reliable quantification of the oligosaccharide content of human milk, providing a reference for determining the specific functions performed by HMOs and at what concentrations they function, as well as for studying the effects of different HMO components and concentrations in human milk on infants.