The catalytic application of a novel ionic liquid and sulfonic acid based bifunctional periodic mesoporous organosilica (BPMO–IL–SO3H) in the one-pot Biginelli reaction was developed. The BPMO–IL–SO3H catalyst was first prepared by hydrolysis and co-condensation of tetramethoxysilane, 1,3-bis(trimethoxysilylpropyl)-imidazolium iodide and 3-mercaptopropyl-trimethoxysilane in the presence of surfactant template under acidic conditions followed by oxidation of SH groups in the presence of hydrogen peroxide. This catalyst was then characterized by thermal gravimetric analysis, transmission electron microscopy, small-angle powder X-ray diffraction, and nitrogen sorption analysis. The performance of the BPMO–IL–SO3H catalyst was investigated in the Biginelli condensation of a variety of different aldehydes with alkylacetoacetates and urea under solvent-free conditions. The catalyst exhibited excellent activity and delivered corresponding Biginelli products in high to excellent yields and selectivities. Moreover, the reactivity, reusability, and stability of the BPMO–IL–SO3H were also investigated under the applied reaction conditions. Our study showed that the catalyst could be recovered and reused at least 10 times without any significant decrease in efficiency.