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Layered double hydroxides are a family of inorganic crystals that have gained a lot of attention due to its special structure and properties such as high porosity, large specific area, and excellent anion exchange ability. In this work, flower‐like NiAl‐layered double hydroxides with high specific area were in situ immobilized onto the stainless steel fibers by bioinspired polydopamine modification method and packed into poly (ether ether) ketone tube for online solid‐phase microextraction with high performance liquid chromatography analysis. Thanks to the high specific surface area and excellent extraction ability of the NiAl‐layered double hydroxides, the fibers showed excellent extraction performance to three Sudan dyes with enrichment factors between 260 to 650 folds. After optimization of the reaction and extraction conditions, an online solid‐phase microextraction method was developed for determination of Sudan dyes in water samples and chili samples. The method has limits of detection of 0.01 to 0.02 ng/mL, good linearity and good reproducibility (≤1.45%). [ABSTRACT FROM AUTHOR]