Previously, we reported a FLucN-LXXLL+LBD-FLucC system that detects VDR ligands using split firefly luciferase techniques, ligand binding domain (LBD) of VDR, and LXXLL sequences that interact with LBD after VDR ligand binding. In vivo , 25-hydroxyvitamin D 3 (25(OH)D 3) and 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3) act as VDR ligands that bind to VDR, and regulate bone-related gene expression. Therefore, the amount of 25(OH)D 3 and 1α,25(OH) 2 D 3 are indicators of bone-related diseases such as rickets and osteoporosis. In this study, we have developed a novel LgBiT-LXXLL+LBD-SmBiT system using NanoLuc Binary Technology (NanoBiT), which has an emission intensity several times higher than that of the split-type firefly luciferase. Furthermore, by using genetic engineering techniques, we attempted to construct a novel system that can specifically detect 1α,25(OH) 2 D 3. Because histidine residues at positions 305 and 397 play important roles in forming a hydrogen bond with a hydroxyl group at position C25 of 25(OH)D 3 and 1α,25(OH) 2 D 3 , His305 and His397 were each substituted by other amino acids. Consequently, the three mutant VDRs, H305D, H397N, and H397E were equally useful to detect 1α,25(OH) 2 D 3 specifically. In addition, among the 58 variants of the LXXLL sequences, LPYEGSLLLKLLRAPVEE showed the greatest increase in luminescence upon the addition of 25(OH)D 3 or 1α,25(OH) 2 D 3. Thus, our novel system using NanoBiT appear to be useful for detecting native vitamin D or its derivatives. • A novel vitamin D biosensor using VDR and nanoluciferase was constructed. • Its emission intensity was several times higher than that using firefly luciferase. • Novel biosensors using VDR mutants specifically detected 1α,25(OH) 2 D 3. • They appear to be useful to measure plasma 1α,25(OH) 2 D 3 concentration. [ABSTRACT FROM AUTHOR]