To screen potential human soluble epoxide hydrolase (hsEH) inhibitors, a high-throughput screening model in 384-well microplate with total volume of 50 μL was established.  Recombinant hsEH  was cloned and expressed in E. coli. and its specific substrate PHOME was synthesized.  The HTS model was based on fluorescence analysis with enhanced sensitivity and specificity (Z ' = 0.65).  A total of 47 360 samples (including 25 040 compounds and 22 320 natural products) were screened, of which 950 samples with inhibition greater than 80% were selected for further rescreening.  Finally, two compounds with high inhibitory activity were identified, whose IC₅₀ value were 8.56 and 4.31 μmol?L⁻¹, separately.  The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.