Ligand-induced conformational changes of nuclear hormone receptors (NRs) are an important start-up of various hormone signaling. However, little is known of the bioanalytical use of the hormone-induced conformational changes of NRs. Here, we describe a generally applicable bioluminescence assay with a genetically encoded bioluminescent indicator to determine androgenicity of ligands based on the intramolecular association of the ligand binding domain of androgen receptor (AR LBD) with the "FQNLF" motif in the N-terminal domain of AR (AR NTD). Firefly luciferase (FLuc) was dissected into N-terminal (1-415 AA) and C-terminal (416-550 AA) fragments. The AR LBD and FQNLF motif of AR NTD were sandwiched between the dissected fragments of FLuc to construct a single molecule-format bioluminescent probe. Androgens induce the association of AR LBD with the FQNLF motif in the NTD, and the subsequent complementation of N- and C-terminal fragments of FLuc partially restores the activities of FLuc. A 10-5 M solution of 5alpha-dihydroxytestosterone (DHT) induced a quick increase in the luminescence intensities from cervical carcinoma-derived HeLa cells carrying the genetic indicator, which reached a plateau in 9 min, whereas DHT withdrawal from the cells by a medium change decreased the luminescence with a slower time course, i.e., approximately 2 h until returning to the background luminescence. The present luminescent indicator was found to exhibit high agonist selectivity and reproducible recovery of the luminescence to a repeated androgen addition and withdrawal. This is the first contribution that cellular signaling steps can be imaged with bioluminescence using a single molecule-format bioluminescence probe (Simbi), in which all the components required for a signal sensing and visualization are integrated. Simbi is applicable to developing biotherapeutic agents effecting to the AR signaling, and for screening adverse chemicals that possibly influence the signal transduction of AR.