To improve the oral bioavailability of quercetin (Q) and achieve colon-specific release, a core sheath electrospun fiber mat containing Q-loaded chitosan nanoparticle (Q-loaded EFM) was developed in this study. The nanoparticle was first fabricated, and its antioxidant activity was as effective as free Q Then the uniform Q-loaded EFM was obtained using response surface methodology optimization, and its core sheath structure was characterized by confocal laser scanning microscopy. In vitro release kinetics confirmed the colon targeting profile, and the release rate of Q varied inversely with fiber diameter. The data of Cell Counting Kit-8 suggested Q-loaded EFM inhibited the proliferation of Caco-2 cells in a dose- and time-dependent manner with an IC50 of 4.36, 2.81, and 2.01 mg/mL after 24, 48, and 72 h, respectively, and it was caused by arresting cell cycle on G(0)/G(1), phase and triggering apoptotic cell death. This study suggests that the Q-loaded EFM represents a promising form in the oral therapy of colon disorders.