Abstract: This study aimed to construct an oral colon-targeted nano-delivery system for the precise delivery of low molecular weight heparin (LMWH), providing a novel strategy for the treatment of ulcerative colitis (UC). Trimethyl chitosan (TMC) was synthesized via one-step methylation reaction. LMWH-loaded TMC nanoparticles (TMC-NPs) were prepared using ionic cross-linking, followed by electrostatic self-assembly coating with sodium alginate (SA) to obtain core-shell structured SA-TMC-NPs. SA-TMC-NPs were found to exhibit a uniform spherical core-shell morphology with a particle size of (216.0±5.8) nm, a Zeta potential of (−35.6±2.3) mV, an entrapment efficiency of (63.7±2.3)%, and a drug-loading capacity of (7.8±0.2)%. Stability studies demonstrated that SA coating significantly enhanced the stability of nanoparticles in simulated gastric fluid and pepsin-containing environments. In vitro release studies revealed that the cumulative release rates of SA-TMC-NPs in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8) were only 6.8% and 22.3%, respectively, significantly lower than those of the uncoated group. Upon entering simulated colonic fluid (pH 7.4), SA coating gradually dissociated, achieving a cumulative release of 56.0% over 24 h, indicating favorable colon-targeted sustained-release properties. The intestinal mucosal permeability experiments showed that TMC-NPs and SA-TMC-NPs could increase the apparent permeability coefficient of LMWH by 3.45-fold and 2.67-fold, respectively. Oral absorption experiments showed that SA-TMC-NPs exhibited sustained-release absorption characteristics, which could reduce the risk of systemic bleeding of LMWH and facilitate colon-targeted delivery. Ex vivo mucoadhesion assays demonstrated that SA-TMC-NPs exhibited significantly higher selective adhesion to inflamed colonic tissues than that to normal colonic tissues; in vivo adhesion experiments showed that SA-TMC-NPs had stronger adhesion to inflamed colonic tissue than TMC-NPs, indicating good inflammation-targeting ability, consistent with the ex vivo results. In conclusion, this study successfully developed an oral nano-delivery system with favorable gastrointestinal stability, pH responsiveness, and inflammation-targeted mucoadhesive properties, providing a novel strategy for the oral application of LMWH in UC treatment.