RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation of RHOA (Ras Homologous family member A). However, when expressed as an intracellular antibody, its blocking effect on RHO signaling led to a loss of actin fibers, which in turn affected cell shape and cell survival. Here, in order to engineer an intracellular biosensor of RHOA-GTP activation, we screened the same phage nanobody library and identified another RHO-GTP selective intracellular nanobody, but with no apparent toxicity. The recombinant RH57 nanobody displays high affinity towards GTP-bound RHOA/B/C subgroup of small GTPases in vitro. Intracellular expression of the RH57 allowed selective co-precipitation with the GTP-bound state of the endogenous RHOA subfamily. When expressed as a fluorescent fusion protein, the chromobody GFP-RH57 was localized to the inner plasma membrane upon stimulation of the activation of endogenous RHO. Finally, the RH57 nanobody was used to establish a BRET-based biosensor (Bioluminescence Resonance Energy Transfer) of RHO activation. The dynamic range of the BRET signal could potentially offer new opportunities to develop cell-based screening of RHOA subfamily activation modulators.