Heparin (HEP) and periodate-oxidized heparin (O-HEP) nanotubes were prepared by combining the template method with a layer-by-layer (LbL) technique. The tubular structure was obtained and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM). O-HEP is one of the HEP derivatives that contains anticoagulant activity and preserves its ability for other effects. Chitosan (CHI) and O-HEP have been used to fabricate nanotubes by covalent cross-linking Schiff base reactions. It is demonstrated that the obtained nanotubes have the significant feature of autofluorescence without the addition of any fluorescent dyes and they retain their anticoagulation activity. Compared with O-HEP/CHI nanotubes, HEP/CHI nanotubes show high anticoagulation activity and do not have autofluorescence. Furthermore, this method could be extended to other copolysaccharide derivatives for the preparation of autofluorescent nanomaterials.