We report the collective alignment of molecular chromophores in the backbone of covalent organic frameworks (COFs) as a new chemical approach to construct efficient two-photon absorption (2PA) materials. Six imine COFs were synthesized from aldehyde-terminated triarylamine linkers and p-phenylenediamine. Attainment of these COFs in single-crystal form revealed the underlying layered structures and precise spatial arrangements of the constituent chromophores including an unusual serrated packing between the layers. Coherent orientation of chromophores within each layer made possible collective alignment of their transition dipoles, while the serrated packing between the layers maximized the crystal anisotropy. As a result, the full potential of molecular chromophores for 2PA was developed. Up to 110-fold enhancement of two-photon action cross-section (2PACS) was achieved for the COFs relative to their constituents, with one member exhibiting a high 2PACS value of 8,756 GM/chromophore-standing among the best molecule-based 2PA materials.