Covalent organic frameworks (COFs) are attractive due to their predictable and tailored periodic frameworks. In the past decades, great efforts have been devoted to crystallinity control, modulation of pores, and so on; however, less attention has been given to atomic-level structural changes. This work provides a fundamental study to further understand the relationship between the periodic structures of the COFs and their unique properties. We explore the relationship of structural isomer linkages in two COFs (TAPT-TFPA and TAPA-TFPT COF). The crystal structures, stacking patterns, pore properties, optical properties, and adsorption properties of these imine-based COFs have been extensively studied and compared. Our results show that compared with TAPA-TFPT COF, TAPT-TFPA COF has superior crystallinity, reversible stacking regulation, and a significant blue shift of UV−vis absorption. These findings provide evidence that monomer pairs must be carefully considered when designing framework materials as these small structural changes can lead to large differences in properties.