The soluble epoxide hydrolase (sEH) is possibly both a marker for and target of numerous diseases. Herein, we describe a homogeneous mix-and-read assay for the detection of human sEH based on using split-luciferase detection coupled with anti-sEH nanobodies. Selective anti-sEH nanobodies were individually fused with NanoLuc Binary Technology (NanoBiT), which consists of a large and small portion of NanoLuc (LgBiT and SmBiT, respectively). Different orientations of the LgBiT and SmBiT-nanobody fusions were expressed and investigated for their ability to reform the active NanoLuc in the presence of the sEH. After optimization, the linear range of the assay could reach 3 orders of magnitude with a limit of detection (LOD) of 1.4 ng/mL. The assay has a high sensitivity to human sEH and reached a similar detection limit to our previously reported conventional nanobody-based ELISA. The procedure of the assay was faster (30 min total) and easy to operate, providing a more flexible and simple way to monitor human sEH levels in biological samples. In general, the immunoassay proposed here offers a more efficient detection and quantification approach that can be easily adapted to numerous macromolecules.