Objective:To establish a high throughput screening (HTS) model for epidermal growth factor (EGF) receptor tyrosine kinase (RTK) inhibitor. Methods: The bioactive EGFR-RTK was expressed by genetic engineering technology. The binding activity and bioactivity of the compounds were examined by surface plasmon resonance and ELISA. Results: The bioactive EGFR-RTK protein was successfully expressed in the prokaryotic expression system and was immobilized on the sensor chip. The equilibrium constants (Kd) between TKI (EI 188) and RTK was 5.00×10-7 mol·L-1 ,and the IC50 was 12.37 μmol·L-1,which was consistent with expected. With this model we screened 31 compounds and found that 6 compounds had binding activity and inhibitory activity. Conclusion: A novel HTS model of EGFR-TKI has been successfully established using surface plasmon resonance biosensor and ELISA, which lays a foundation for discovery of new TKIs.