S-layer proteins (Slps) are crystalline arrays of protein on bacterial cell surface layers. Owning to their capability to reassemble on the surface of lipid layers, Slps have been employed to modify liposomes for various profits. But the interaction information between Slps and positively charged liposomes are destitute, especially the gastrointestinal adhesion of Slps-coating liposomes is rarely reported. In the present work, the Slps extracted from Lactobacillus helveticus were reassembled on the surface of novel positively charged liposomes composed of soybean lecithin, Eudragit®RL100 and cholesterol. The particle size and remarkable changes of Zeta potential with various Slps/lipid weight ratios were determined by dynamic light scattering and phase analysis light scattering. Significant difference in fluorescence dequenching percentage of liposomes decorated by ODA-FITC confirmed Slps self-reassemble on the surface of liposomes. A higher integrity of vesicular membrane after the addition of Triton X-100 solution demonstrated the stability enhancement of Slps-coating liposomes. Fourier transform infrared (FTIR) spectroscopy illustrated the interaction came from non-covalent bond. The mucoadhesion of Slps-coating liposomes was evaluated by the resident FITC-LP on the gastric and intestinal tract of mice at 7 h and 12 h after intragastrical administration, which proved that the Slps-coating improved the gastrointestinal adhesion significantly.