Due to their permanent porosity, robust chemical stability, and tunable structure, covalent organic frameworks (COFs) are very attractive in the application of energy storage, catalysis, sorption, and sensing. However, the very low conductivity of COFs severely restricts their application in electrochemical sensing. Here, an aza-fused π-conjugated COFs with abundant carbonyl groups (COF1) was synthesized and deployed as electrode materials in electrochemical sensing for the first time. The current response of the acetylcholinesterase biosensor based on COF1 increases three times when compared to the electrode without COF1. The effects of carbonyl groups on signal enhancement were proved in depth by a series of characterization and comparison experiments with the prepared COF2 without carbonyl groups. The results demonstrated that exposed carbonyl active sites of COF1 can promote the effective immobilization and bioactivity preserving of enzyme molecules and contribute to the enrichment of analytes. Together with the good conductivity of COF1 derived from a fully extended 2D aromatized π-conjugated system, all of which improve the biosensor performance. The COF1-based biosensor exhibited fast response speed, high sensitivity, good selectivity and practicability, and robust stability for organophosphorus pesticide detection and proved to be a promising tool for the rapid and onsite detection of organophosphorus pesticides in the environment.