Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling performance has not been systematically characterized. Through concurrent side scattering and fluorescence detection of single EVs as small as 40 nm in diameter by a laboratory-built nano-flow cytometer (nFCM), present study identified that (1) PKH67 and PKH26 could maximally label ∼60%-80% of EVs isolated from the conditioned cell culture medium (purity of ∼88%) and ∼40%-70% of PFP-EVs (purity of ∼73%); (2) excessive PKH26 could cause damage to the EV structure; (3) di-8-ANEPPS and high concentration of DiI could achieve efficient and uniform labelling of EVs with nearly 100% labelling efficiency for di-8-ANEPPS and 70%-100% for DiI; (4) all the four tested LMDs can aggregate and form micelles that exhibit comparable side scatter and fluorescence intensity with those of labelled EVs and thus hardly be differentiate from each other; (5) as the LMD concentration went up, the particle number of self-aggregates increased while the fluorescence intensity of aggregates remained constant; (6) PKH67 and PKH26 tend to form more aggregated micelles than di-8-ANEPPS and DiI, and the effect of LMD self-aggregation can be negligible at optimal staining conditions. (7) All the four tested LMDs can label almost all the very-low-density lipoprotein (VLDL) particles, indicating potential confounding factor in plasma-EV labelling. Besides, it was discovered that DSPE-PEG2000 -biotin can only label ∼50% of plasma-EVs. The number of LMP inserted into the membrane of single EVs was measured for the first time and it was confirmed that membrane labelling by lipophilic dyes did not interfere with the immunophenotyping of EVs. nFCM provides a unique perspective for a better understanding of EV labelling by LMD/LMP.