Biofilm is an extracellular matrix of bacteria and serves as a protective shield of bacterial communities. It is crucial for microbial growth and one of the leading causes of human chronic infections as well. However, the structures and molecular mechanism of biofilm formation remain largely unknown. Here, we examined a protein, BslA, expressed in the biofilms of Bacillus subtilis. We characterized the Langmuir monolayers of BslA at the air/water interface. Using techniques in surface chemistry and spectroscopy, we found that BslA forms a stable and robust Langmuir monolayer at the air/water interface. Our results show that the BslA Langmuir monolayer underwent two-stage elasticity in the solid state phase upon mechanical compression: one is possibly due to the intermolecular interaction and the other is likely due to both the intermolecular compulsion and the intramolecular distortion. The Langmuir monolayer of BslA shows abrupt changes in rigidities and elasticities at similar to 25 mN/m. This surface pressure is close to the one at which BlsA saturates the air/water interface as a self-assembled film without mechanical compression, corresponding to a mean molecular area of similar to 700 angstrom(2) per molecule. Based on the results of surface UV-visible spectroscopy and infrared reflective-absorption spectroscopy, we propose that the BslA Langmuir monolayer carries intermolecular elasticity before similar to 25 mN/m and both intermolecular and intramolecular elasticity after similar to 25 mN/m. These results provide valuable insights into the understanding of biofilm-associated protein under high mechanical force, shedding light on further investigation of biofilm structure and functionalities.