The study presents a novel, two-step aqueous method for immobilizing biomolecules onto surfaces, inspired by mussel adhesive proteins. The method involves dip-coating a biomimetic polymer thin film (polydopamine) onto a substrate and then conjugating biomolecules to this film. Polydopamine, a polymer that forms at alkaline pH, exhibits latent reactivity towards nucleophiles and is resistant to hydrolysis, making it suitable for biomolecule immobilization. The approach is demonstrated through the immobilization of trypsin onto various substrates, including organic and inorganic materials, without the need for surface pretreatment. X-ray photoelectron spectroscopy (XPS) analysis confirms the successful formation of polydopamine films and the subsequent immobilization of trypsin. The method is also shown to be pH-sensitive, allowing for the discrimination between different nucleophiles, such as amines and imidazoles, through pH control. This technique offers a versatile and efficient way to immobilize biomolecules, potentially useful in various biological and physical applications.The study presents a novel, two-step aqueous method for immobilizing biomolecules onto surfaces, inspired by mussel adhesive proteins. The method involves dip-coating a biomimetic polymer thin film (polydopamine) onto a substrate and then conjugating biomolecules to this film. Polydopamine, a polymer that forms at alkaline pH, exhibits latent reactivity towards nucleophiles and is resistant to hydrolysis, making it suitable for biomolecule immobilization. The approach is demonstrated through the immobilization of trypsin onto various substrates, including organic and inorganic materials, without the need for surface pretreatment. X-ray photoelectron spectroscopy (XPS) analysis confirms the successful formation of polydopamine films and the subsequent immobilization of trypsin. The method is also shown to be pH-sensitive, allowing for the discrimination between different nucleophiles, such as amines and imidazoles, through pH control. This technique offers a versatile and efficient way to immobilize biomolecules, potentially useful in various biological and physical applications.