Previous our work suggested that V1b receptor for neuropituitary hormone vasopressin enhances cellular signal leading to morphine tolerance by functionally interacting with mu-opioid receptor (MOR) and beta-arrestin 2. However, it has not been clarified how these three signaling molecules are arranged. Here, we explored a possibility of formation of three-molecule complex using bioluminescence resonance energy transfer (BRET) among split nanoluciferase and Venus fluorescent protein. To this end, two pieces of a split nanoliciferase were connected to carboxyl termini of V1b or MOR, and Venus to beta-arrestin2. We successfully detected BRET between V1b-MOR heteromer receptor and beta-arrestin 2-Venus. Importantly, this interaction was enough high at basal, indicating constitutive interaction without agonist. Furthermore, unlike the receptor tagged with a whole nanoluciferase, BRET signal from interaction among V1b-MOR heteromer with split nanolucifarese and beta-arrestin 2-Venus increased further after agonist treatment. These results indicated that we successfully developed screening method to search for a chemical that could suppress morphine tolerance by acting at V1b receptor in the V1b-MOR heteromer.