Although magnolol (Mag), an anti-inflammatory natural compound, has been demonstrated to play protective effects on ulcerative colitis (UC), its application as an alternative therapeutic reagent for UC treatment is still greatly impeded due to its poor stability in the gastrointestinal tract and insufficient accumulation in the inflamed colon lesion. Nano-/microsized drug delivery systems can potentially overcome some challenges regarding the oral administration of phytochemicals, which still confront premature early drug release, degradation of NPs, or the sustained drug release of MPs. In this study, we primarily loaded Mag into the core-shell zein-based nanoparticles with chondroitin sulfate coating (Mag@CS-Zein NPs) with an average size of 142.27 ± 5.11 nm, showing significant macrophage-targeting and enhanced colon epithelial cellular uptake capacity. Then, we embedded Mag@CS-Zein NPs into hydrogel microspheres via an electrospraying technology. The Mag@CS-Zein NPsinMPs presented a uniform-sized sphere with an average size of 164.36 ± 6.29 μm and sustained drug-release profiles. Compared to CS-Zein NPs, the developed CS-Zein NPsinMPs exhibited prolonged colon retention on the inflammatory surface, as seen from ex vivo and in vivo imaging fluorescence adhesion experiments. Based on the advantage of the combination of hybrid nanoparticles-in-microparticles, oral administration of Mag@CS-Zein NPsinMPs significantly alleviated colitis symptoms in DSS-treated mice by regulating the expression levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) and anti-inflammatory cytokines (IL-10) and factor accelerated colonic mucosal barrier repair via upregulating the expression of ZO-1 and occludin. This study provides great insights into the oral drug delivery of natural compounds for UC therapy.