Single-particle tracking (SPT) is promising for precise analysis of spatiotemporal dynamics of proteins in living cells. Nonspecific fluorescent labeling and photobleaching of fluorescence in conventional SPT methods disturb reliable analysis. Here we aimed to develop a SPT technique for precise analysis of protein dynamics in living cells. We employed a regenerative fluorescence labeling technique named DeQODE technology. In this technology, a small probe molecule consisting of a fluorescent moiety and a quencher moiety is designed to reversibly bind to a protein tag named DeQODE tag which is based on a single-chain antibody against the quencher moiety. Binding of the small probe molecule to DeQODE tag turns on fluorescence. Repeated binding and dissociation of the probe to the intracellularly expressed DeQODE tag fused with the target protein in the presence of low-concentration probe molecule enables long-term SPT. To demonstrate the proof of the concept, we performed SPT of syntaxin 1A, a component of the SNARE complex, in COS7 cells, INS-1 cells, and cultured hippocampal neurons. In these cells expressing DeQODE tag-fused syntaxin 1A, we successfully obtained SPT data for hours. These results suggest that the DeQODE-based SPT is useful for precise molecular dynamics analysis in physiological and pharmacological responses.