<p>The efficient enrichment and simultaneous detection of trace levels of angiotensin (Ⅰ, Ⅱ, Ⅲ) and aldosterone in plasma pose certain technical challenges. Herein, we propose a novel strategy where a cationic covalent organic framework (Py-BPy²⁺-COF) was served as an attractive adsorption material for efficient enrichment of angiotensin (Ⅰ, Ⅱ, Ⅲ) and aldosterone. Adsorption experiments and model fitting revealed that Py-BPy²⁺-COF exhibited adsorption capacities ranging from 5.02 to 26.12&#xa0;μg g⁻¹ for the four target analytes. The Hirshfeld partition analysis elucidated that the interaction forces of angiotensin and aldosterone with Py-BPy²⁺-COF originated from electrostatic interactions, π-π stacking and hydrophobic interactions. Py-BPy²⁺-COF was employed as the sorbent packing in a 96-well plate coupled with liquid chromatography-tandem mass spectrometry, achieving high sensitivity, excellent reproducibility, high recoveries (80.1%-94.9%), low limits of detection (5–50&#xa0;pg mL⁻¹), and cost-effectiveness, while maintaining high extraction efficiency even after 10 repeated uses. This study provides a novel approach for the detection of angiotensin (Ⅰ, Ⅱ, Ⅲ) and aldosterone in plasma, and demonstrates the potential of cationic COFs as functional materials in biomedical analysis.</p> Graphical Abstract <p></p>