b-Series gangliosides are abundant in central nervous tissues and are involved in important nerve processes. However, their functions are complicated because of their properties of forming dynamic domains in cell plasma membranes (PMs), called lipid rafts. In this study, we aim to develop fluorescently labeled b-series gangliosides that are useful for single-molecule imaging. The chemical synthesis of fluorescent GD3 and GQ1b was achieved using sialylation and ganglioside synthetic methods previously developed by our group. Furthermore, biophysical evaluations demonstrated that synthesized fluorescent GD3 and GQ1b behaved as raft molecules on cell PMs, suggesting their applicability to the study of raft-associated interactions.