Enzyme-instructed self-assembly is an increasingly attractive topic owing to its broad applications in biomaterials and biomedicine. In this work, we report an approach to construct enzyme-responsive aqueous surfactant two-phase (ASTP) systems serving as enzyme substrates by using a cationic surfactant (myristoylcholine chloride) and a series of anionic surfactants. Driven by the hydrophobic interaction and electrostatic attraction, self-assemblies of cationic-anionic surfactant mixtures result in biphasic systems containing condensed lamellar structures and coexisting dilute solutions, which turn into homogeneous aqueous phases in the presence of hydrolase (cholinesterase). The enzyme-sensitive ASTP systems reported in this work highlight potential applications in the active control of biomolecular enrichment/release and visual detection of cholinesterase.