As a new type of crystalline porous polymer materials, covalent organic frameworks (COFs) with their unique features such as large surface area, tunable pore sizes, strong π-π stacking effect and size exclusion effects, have attracted wide attention in the analytical field. Due to the lack of catalytically active metal centers in bare COFs, functionalized COFs that are hybridized or modified with nanomaterials improve reactive activation and show better analytical performance for a variety of detection scenarios with complex analytes. Herein, we focused on the functionalized COFs used in bioanalysis ranging from nucleic acids, peptides, and proteins, to microorganisms, and discussed the functionalization strategy and unique structures and properties applied in the different stages of biosensing and advantages compared to other hybrid materials. Finally, challenges and future research directions of functionalized COFs in bioanalysis are discussed.