Abstract: 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). N,N,N-Trimethyl chitosan (TMC) was synthesized via a one-step methylation reaction and characterized by ¹H NMR and FTIR. 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. The physicochemical properties, stability under various conditions, in vitro drug release behavior, and ex vivo mucoadhesive capacity of the nanoparticles were systematically evaluated. Results showed that TMC with a quaternization degree of 70% was successfully synthesized. SA-TMC-NPs exhibited 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 of (7.8 ± 0.2)%. Stability studies demonstrated that the 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), the SA coating gradually dissociated, achieving a cumulative release of 56.0% over 24 h, indicating favorable colon-targeted sustained-release properties. Ex vivo mucoadhesion assays demonstrated that SA-TMC-NPs exhibited significantly enhanced selective adhesion to inflamed colonic tissues, with an adhesion rate of 36.46%, higher than that to normal colonic tissues (26.96%). In conclusion, this study successfully developed an oral nano-delivery system with pH responsiveness, favorable gastrointestinal stability, and inflammation-targeted mucoadhesive properties, providing a novel strategy for the oral application of LMWH in UC treatment.