Through a dual‐ligand synthetic approach, five isoreticular primitive cubic (pcu)‐type pillared‐layer metal–organic frameworks (MOFs), Zn2(dicarboxylate)2(NI‐bpy‐44)⋅x DMF⋅y H2O, in which dicarboxylate=1,4‐bdc (1), Br‐1,4‐bdc (2), NH2‐1,4‐bdc (3), 2,6‐ndc (4), and bpdc (5), have been engineered. MOFs 1–5 feature twofold degrees of interpenetration and have open pores of 27.0, 33.6, 36.8, 52.5, and 62.1 %, respectively. Nitrogen adsorption isotherms of activated MOFs 1′–5′ at 77 K all displayed type I adsorption behavior, suggesting their microporous nature. Although 1′ and 3′–5′ exhibited type I adsorption isotherms of CO2 at 195 K, MOF 2′ showed a two‐step gate‐opening sorption isotherm of CO2. Furthermore, MOF 3′ also had a significant influence of amine functions on CO2 uptake at high temperature due to the CO2–framework interactions. MOFs 1–5 revealed solvent‐dependent fluorescence properties; their strong blue‐light emissions in aqueous suspensions were efficiently quenched by trace amounts of nitrobenzene (NB), with limits of detection of 4.54, 5.73, 1.88, 2.30, and 2.26 μm, respectively, and Stern–Volmer quenching constants (Ksv) of 2.93×103, 1.79×103, 3.78×103, 4.04×103, and 3.21×103 m−1, respectively. Of particular note, the NB‐included framework, NB@3, provided direct evidence of the binding sites, which showed strong host–guest π–π and hydrogen‐bonding interactions beneficial for donor–acceptor electron transfer and resulting in fluorescence quenching. Space for guests: Five isoreticular luminescent pillared‐layer metal–organic frameworks showing CO2 uptake and the sensitive detection of nitrobenzene (NB) in aqueous solution are engineered. The NB‐included framework provides direct evidence for framework–NB interactions.