The split-and-pool method has been widely used to synthesize chemical libraries of a large size for early drug discovery, albeit without the possibility of meaningful quality control. In contrast, a self-assembled DNA-encoded chemical library (DEL) allows us to construct an m x n-member library by mixing an m-member and an n-member pre-purified sub-library. Herein, we report a trio-pharmacophore DEL (T-DEL) of m x l x n members through assembling three pre-purified and validated sub-libraries. The middle sub-library is synthesized using DNA-templated synthesis with different reaction mechanisms and designed as a linkage connecting the fragments displayed on the flanking two sub-libraries. Despite assembling three fragments, the resulting compounds do not exceed the up-to-date standard of molecular weight regarding drug-likeness. We demonstrate the utility of T-DEL in linker optimization for known binding fragments against trypsin and carbonic anhydrase II and by de novo selections against matrix metalloprotease-2 and −9.