As novel food nutritional fortifiers, human milk oligosaccharides (HMOs) have attracted widespread attention regarding the accuracy of their detection methods. To optimize the high-performance liquid chromatography (HPLC) method for seven HMOs, the effects of sample preparation and instrumental parameters on their qualitative and quantitative analysis were investigated in this study. After redissolution and dilution with water, 11 types of food samples were treated with starch glucosidase and β-galactosidase for purification. The supernatants were collected after fluorescent labeling. The separation was performed using acetonitrile and NH4COOH (50 mmol·L-1, pH 4.2 ± 0.1) as the mobile phases. The excitation and emission wavelengths were 330 nm and 420 nm, respectively. The quantification was carried out using laminaritriose as an internal standard. The linear correlation of 2’-fucosyllactose was up to 0.99999 within the range of 5-500 mg·L-1, and that of the other 6 HMOs was up to 0.99999 within the range of 1.5-150 mg·L-1. The recovery rate was 90.62%-109.71%, and the relative standard deviation was less than 5.23%. The limits of detection (LOD) of the 7 HMOs were 2.00 mg/100g (solid) and 0.20 mg/100g (liquid), and the limits of quantitation (LOQ) were 5.00 mg/100g (solid) and 0.50 mg/100g (liquid), respectively. This method is not applicable to the determination of difucosyllactose in foods containing isomaltose oligosaccharide. Using the optimized method, 222 batches of commercially infant formula and special medical foods were tested. Currently, most of them are mainly supplemented with 2’-fucosyllactose and lacto-N-neotetraose, while lacto-N-neotetraose was detected at a relatively high frequency. This method has a simple pretreatment process, high sensitivity, good repeatability. It can provide a theoretical foundation for the formulation of the subsequent national standards.