Polydiacetylenes provide a good biological/chemical label-free detection system owing to their blue to red color change and fluorescence change from non-fluorescence to red fluorescence. One of the important factors to consider before applying polydiacetylenes to various sensors is to enhance the sensitivity against specific molecules. This paper reports two methods for stacking polydiacetylene vesicles for fluorescence signal enhancement of polydiacetylene on solid substrates. This focuses on how to achieve a highly sensitive sensor chip by introducing two strategies to immobilize the vesicles effectively on substrates. One method is layer-by-layer deposition through reactions of vesicles and avidins alternately on an avidin treated substrate. The other is to prepare a clustered vesicles solution by mixing the appropriate amount of a biotinylated vesicle solution and avidin solution, and then immobilize the clustered vesicles on the substrates. The former allows easy control of the layer thickness, whereas the latter can shorten the process time. These strategies can be applied to a range of biological/chemical sensors for fluorescence signal enhancement.