Abstract: Objective: To screen and optimize the method for preparing three-dimensional structural samples of restriction endonuclease Bsa I. Methods: In this study, the Escherichia coli expression system was employed to express the Bsa I protein and its selenomethionine derivative. Firstly, a recombinant expression vector pBAD-Bsa I was constructed and transformed into Escherichia coli (E.coli) ER2566 for expression. Purification was carried out using affinity chromatography and anion exchange chromatography. Subsequently, selenomethionine derivation situation was assessed using mass spectrometry and circular dichroism spectroscopy, followed by enzyme activity determination. Lastly, preliminary crystal growth studies were conducted using the sitting-drop method. Results: Through a two-step purification approach, recombinant Bsa I and Se-Bsa I selenomethionine derivative with a purity exceeding 90% were obtained. Mass spectrometry analysis revealed that all 11 methionine residues in the recombinant Se-Bsa I protein were selenomethionine-incorporated. Circular dichroism spectroscopy and enzyme activity testing confirmed that the selenomethionine incorporation had no significant impact on the structure and activity of Bsa I protein. Crystallization experiments demonstrated that the recombinant Bsa I protein could not only form granular crystals under conditions of 0.2 mol/L magnesium acetate tetrahydrate at pH6.5, 0.1 mol/L sodium cacodylate trihydrate with 20% polyethylene glycol 8000, but also form spherical structures under conditions of 0.1 mol/L sodium acetate trihydrate at pH4.6 and 2 mol/L ammonium sulfate. Conclusion: This study successfully achieved the recombinant expression of Bsa I and Se-Bsa I selenomethionine derivative, conducted preliminary screening of protein crystal conditions, aiming to provide valuable insights for deciphering the three-dimensional structure of Bsa I protein.