We report a fluorescence-based approach to probing the conformation of a macromolecule, poly(allylamine hydrochloride) (PAH), in bimodal mesoporous silica (BMS) particles. The method involves monitoring the fluorescent properties of the probe, 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (4-PSA), upon electrostatic binding to PAH molecules adsorbed in the nanopores of the BMS particles. PAH infiltration into the BMS particles, quantified by thermogravimetric analysis and visualized by confocal laser scanning microscopy, was examined as a function of PAH adsorption time, PAH molecular weight, and the sodium chloride (NaCl) concentration and pH of the PAH adsorption solution. The conformation of PAH molecules in the nanopores was investigated by incubating the PAH-loaded BMS particles in 4-PSA and using the ratio of the excimer to monomer emission intensity to discern differences in the PAH conformation in the nanopores. Control experiments involving nonporous silica (NS) particles were also conducted to determine the extent to which the nanopores within the BMS particles influence the degree of PAH adsorption and the conformation of the adsorbed PAH molecules. The data indicate that PAH molecules adsorbed in the nanopores adopt a more coiled conformation than PAH molecules adsorbed on NS particles over a wide range of conditions. Further, the conformation of PAH molecules in the nanopores can be tuned by adjusting the NaCl concentration and/or pH of the PAH adsorption solution. 4-PSA titration experiments revealed that at saturation binding there are ca. 3.8 PAH monomer units per 4-PSA molecule. This study provides insights into macromolecule infiltration and conformation in nanopores, which are important for the application of mesoporous materials in the fields of adsorption/immobilization, catalysis, delivery, sensing, separations, and synthesis.