In this paper, the size effect of the polydiacetylene vesicles functionalized with glycolipids on their colorimetric detection ability has been studied. Polydiacetylene vesicles, in which were incorporated glycolipids acted as a model system for the affinochromatic property. Visible color changes from blue to red could be observed to the naked eye owing to Con A binding to the sugar moiety and be detected quantitatively by the visible absorption spectrum. In the experiment, small and uniform vesicles were obtained after extrusion through membranes with different pore sizes. The morphology and mean size distribution of the extruded vesicles were studied by transmission electron microscopy (TEM) and dynamic light scattering (DLS), respectively. Our work shows that the smaller the vesicles are, the stronger is the effect, making the detection of Con A easier. The results may apply to the sensitivity enhancement of polydiacetylene biosensors for the recognition of other biomolecules.