A facile method for immobilizing biomolecules onto surfaces using polydopamine coatings is described. The approach involves dip-coating a substrate with a polydopamine film, followed by conjugation of biomolecules to the film. Polydopamine, derived from dopamine, forms a thin, adherent film that is reactive towards nucleophiles and is resistant to hydrolysis in aqueous environments. This method allows for selective conjugation based on the pKa of nucleophiles and is effective for a wide range of substrates, including organic and inorganic materials. The polydopamine film is formed by immersing substrates in an alkaline dopamine solution, leading to the formation of a reactive film. This film can then be used to covalently immobilize biomolecules through reactions with nucleophiles. For example, trypsin was successfully immobilized on various substrates, including copper, titanium oxide, polycarbonate, and cellulose. XPS analysis confirmed the presence of polydopamine on these substrates and the successful conjugation of trypsin. The method is highly effective and stable, even under alkaline conditions, which is a significant advantage over traditional methods like NHS esters that are prone to hydrolysis. The polydopamine coating also allows for pH-dependent control of biomolecule immobilization, enabling selective conjugation based on the pKa of the biomolecules. This was demonstrated using a model peptide, His-EG3-Lys, where immobilization was pH-dependent due to the different pKa values of histidine and ε-amino groups. The study highlights the versatility and stability of polydopamine coatings for biomolecule immobilization, offering a simple and effective alternative to traditional methods. The method is applicable to a wide range of materials without surface pretreatment and is resistant to hydrolysis, making it a promising approach for various biomedical and engineering applications.A facile method for immobilizing biomolecules onto surfaces using polydopamine coatings is described. The approach involves dip-coating a substrate with a polydopamine film, followed by conjugation of biomolecules to the film. Polydopamine, derived from dopamine, forms a thin, adherent film that is reactive towards nucleophiles and is resistant to hydrolysis in aqueous environments. This method allows for selective conjugation based on the pKa of nucleophiles and is effective for a wide range of substrates, including organic and inorganic materials. The polydopamine film is formed by immersing substrates in an alkaline dopamine solution, leading to the formation of a reactive film. This film can then be used to covalently immobilize biomolecules through reactions with nucleophiles. For example, trypsin was successfully immobilized on various substrates, including copper, titanium oxide, polycarbonate, and cellulose. XPS analysis confirmed the presence of polydopamine on these substrates and the successful conjugation of trypsin. The method is highly effective and stable, even under alkaline conditions, which is a significant advantage over traditional methods like NHS esters that are prone to hydrolysis. The polydopamine coating also allows for pH-dependent control of biomolecule immobilization, enabling selective conjugation based on the pKa of the biomolecules. This was demonstrated using a model peptide, His-EG3-Lys, where immobilization was pH-dependent due to the different pKa values of histidine and ε-amino groups. The study highlights the versatility and stability of polydopamine coatings for biomolecule immobilization, offering a simple and effective alternative to traditional methods. The method is applicable to a wide range of materials without surface pretreatment and is resistant to hydrolysis, making it a promising approach for various biomedical and engineering applications.