Three-dimensional (3D) covalent organic frameworks (COFs) are crystalline porous polymers with potential in numerous high-tech applications, but the linkages involved in their synthesis are still rather limited. Herein we report the first case of 3D sp2 carbon-linked COFs fabricated by the formation reaction of C=C bonds and their application in bioresponsive fluorescence imaging. These new COFs, namely JUC-580 and JUC-581, showed high stability and excellent light-emitting properties in solid state and dispersed in various solvents. Furthermore, we investigated the potential application of JUC-581 for a drug carrier combined with bioresponsive fluorescence imaging. The results indicated that 3D sp2 carbon-linked COFs are not only potential drug-loaded and sustained release materials but also promising cell fluorescent stains. This study expands the structural categories of 3D COFs based on different linkages, and promotes their prospective applications for biomedicine and fluorescent materials.