To screen potential human soluble protein tyrosine phosphatase 1B (PTP1B) inhibitors, a high-throughput screening (HTS) model in 384-well microplate with total volume of 50 μL was established.  Recombinant PTP1B was cloned and expressed in E. coli. with its specific substrate 4-nitrophenyl phosphate disodium salt hexahydrate (PNPP).  The HTS model was based on enzyme reaction rate with enhanced sensitivity and specificity (Z' = 0.78).  A total of 24 240 samples were screened, among them 80 samples with inhibition greater than 70% were selected for further rescreening.  Finally, six compounds with high inhibitory activity were identified, whose IC₅₀ values were 21.58, 18.39, 15.37, 11.92, 37.27, and 36.61 μg?mL⁻¹, separately.  The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.