Unlike other extracellular vesicle (EV) subtypes such as exosomes, the lack of well-defined universal markers on the surface of microvesicles (MVs) has led to difficulty in the detection of the entire MV population. To design a universal MV detection method, we reported highly sensitive electrical detection of MVs using a reduced graphene oxide (RGO)-based field-effect transistor (FET) biosensor by the introduction of a membrane biotinylation strategy in this work. Biotinylated MVs (B-MVs) were obtained by supplying the culture medium with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG-biotin) while cultivating the cells. Excellent biotinylation efficiency of MVs (92.6%) was then realized. A streptavidin (SA) probe was subsequently modified onto the channel surface of the as-fabricated RGO-based FET device, which was capable of specifically recognizing B-MVs due to the high affinity between SA and biotin in a 1 : 4 recognition format. The results showed that the RGO-based FET biosensor could detect B-MVs in a wide range from 105 particles per mL to 109 particles per mL with a low detection limit down to 20 particles per μL, which was the lowest value compared with other previously reported results. This platform also allowed distinguishing B-MVs from other unbiotinylated EV types such as MVs and exosomes, exhibiting excellent specificity. Moreover, this FET biosensor demonstrated the capability of detecting B-MVs derived from different cell lines including cancer cells and normal cells, indicating its versatility and potential applications in the biomedical field.