By using progesterone as template molecule, methacrylic acid (MAA) as monomer, ethylene glycol dimethacrylate (EGDMA) as cross-linking agent and benzophenone (BP) as photo-initiator, the molecularly imprinted film (MIF) was synthesized by in-situ photo-grafting. The chip surface modified with aprogesterone - imprinted film was characterized by atomic force microscopy (AFM), showing that many cavities at nanometer scales were dispersed on the surface of MIF. By using surface plasmon resonance technology, a good linear relationship was obtained between the reflectivity and progesterone concentrations arranging from 1.0 to 1.0 ×103 pmol/ L, and the detection limit was 0.3 pmol/ L. The MIF had good selectivity for template molecules. The MIF sensor could be regenerated 7 times of rinsing and re-adsorption without significant deterioration of the sensor performance. After placed for 90 days, the MIF still performed good rinsing and adsorption, indicating that it had good repeatability and stability. Progesterone was detected in artificial urine with recoveries in the range of 96.0% -102.5%. © 2021, Youke Publishing Co.,Ltd. All rights reserved.