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Immunofluorescence (IF) staining represents a convenient and cost-effective approach to analysing single extracellular vesicles (EVs) and identifying subpopulations with specific roles or biological functions. However, the application of the method is challenged by the weak and unstable signals generated by the low abundant markers carried by the vesicles. In this study, we report the development of an IF strategy based on tyramide signal amplification (TSA) that employs tyramide probes for signal enhancement. The technique is first validated on glioblastoma circulating tumour cells (GBM CTCs) and systematically compared with conventional approaches using fluorescently labelled primary and secondary antibodies. Thereafter, the proposed method is adapted, tested and optimised for the multiplexed fluorescent staining of single EVs isolated from the parental GBM CTCs. The results demonstrate specific staining of single EVs by the developed TSA method, highlighting its advantages of amplified (>6×) signal intensities, more stable signals and broader (∼3×) signal dynamic ranges as compared to the conventional fluorescence methods. The developed protocol also supports multiplexing by incorporating a quenching buffer between the different staining colours. Finally, the protocol demonstrates its applicability to CTCs and EVs derived from plasma samples of GBM patients, with easy adaptation to other cancers or proteins of interest.