Chiral separation plays a pivotal role in both practical applications and industrial productions. However, traditional chiral stationary phases (CSPs) exhibit inherent instability in alkaline environments, presenting a significant challenge despite their importance. Herein, basophilic alcalase is creatively developed to fabricate ultrastable protein-based CSPs that can efficiently work under alkaline conditions. An in-depth theoretical simulation is conducted to unveil the unique three-dimensional conformation of alcalase, showing selective affinity towards various enantiomers of chiral amino acids and drugs, especially acidic substrates. Subsequently, an in situ assembly strategy is used to immobilize alcalase within a hydrazone-linked covalent organic framework (COF) platform. The generated protein-based CSPs enable successful baseline separation (R