Abstract: A method was developed for the simultaneous determination of fat-soluble vitamins A, D₂, D₃ and α-, β-, γ-, δ-vitamin E in children's formula milk powder by combining online solid phase extraction (SPE) and two-dimensional liquid chromatography. The saponified samples were directly injected into the constructed system and purified by using the polymeric-based PLRP-S column as the SPE column. Then the target analytes were eluted in back-flush mode from the SPE column by SPE pump and refocused on the head of the first-dimensional column by using online dilution. Vitamins A and four E homologues were separated on a NuovaSil^TM PFP column (4.6 mm×150 mm, 3 μm) as the first-dimensional column using a mobile phase consisting of methanol and 0.1% formic acid in water by gradient elution, and vitamins D₂ and D₃ were separated on a ChromCore PAH column (4.6 mm×150 mm, 3 μm) as the second-dimensional column using a mobile phase consisting of methanol and acetonitrile (90:10, v/v). The results showed good linearity in the range of 5.0~500.0 ng/mL for vitamin A, 1.0~100.0 ng/mL for vitamin D and 50.0~5000.0 ng/mL for vitamin E, and the correlation coefficients were all better than 0.9999. The limits of quantification for vitamins A, D₂, D₃ and α-, β-, γ-, δ-vitamins E were 5.11, 1.22, 1.25, 83.82, 57.27, 56.61, 54.82 µg/100 g, respectively. The spiked recoveries were 91.20%~104.47%, and the relative standard deviations were 0.94%~3.34%. The results show that the method established in this paper has the advantages of simple pretreatment, high automation, high analytical efficiency, high accuracy and good reproducibility, and is suitable for the determination of vitamins A, D and E in children's formula milk powder.