Aim To prepare chitosan-coated liposomes of fluorouracil for colon targeting and investigate their morphology and in vitro release. Methods Rhodamine B isothiocyanate and Bodipy-PC were used to label chitosan and the liposomes, respectively. Laser confocal scanning microscopy was used to explore the structure of chitosan-coated liposomes, and the release of chitosan-coated liposomes in simulated gastric solution, simulated enteric fluid and simulated colon fluid were investigated. Results The size of liposomes was measured by laser diffraction before and after polymer coating. The mean diameter of liposomes was in the range of 1.43-3.82 μm, whereas after coating 2.06-5.62 μm. For three types of liposomes, negatively charged, positively charged and neutrally charged, the entrapment rate was 99%, 61%, and 72%, respectively. The cumulative release of chitosan-coated liposomes in simulated gastric fluid was only 6.28% in 4 h, and in simulated enteric fluid only 6.8% in 8 h. But in the simulated colon fluid contained β-glucosidase the release was rapidly increased to a t 1/2 of 3.63 h. Conclusion The preparation technology of chitosan-coated liposomes for colon targeting was feasible. In vitro release in the simulated colon fluid was fitted to Higuchi equation.