A method was developed for the simultaneous determination of six human milk oligosaccharides (HMOs) in infant formula using high performance liquid chromatography-fluorescence detector (HPLC-FLD). The samples were first enzymatically digested and precipitated with organic solvents to remove interfering substances, and then separated using a hydrophilic chromatographic column. Under the optimal conditions, the six HMOs showed good linearity in their respective concentration ranges, with determination coefficients (R²) above 0.999. The recoveries of the spiked samples ranged from 95.91% to 105.03%, with relative standard deviations (RSD) of less than 2%. The limits of detection (LOD) and quantification (LOQ) were in the range of 0.0036–0.0064 and 0.0120–0.0214 g/100 g, respectively. The developed method is characterized by high sensitivity and good stability, and can meet the requirements of the determination of the six HMOs in infant formula.