Polyelectrolyte-nanoparticle hybrid multilayer films were used as templates for the preparation of nanoporous polymer thin films. The hybrid multilayers were constructed by consecutively depositing an anionic blend of silica nanoparticles (SiO2 NPs) and poly(acrylic acid) (PAA) and a cationic polyelectrolyte, poly(allyamine hydrochloride) (PAH). Layer-by-layer growth of the multilayers with different SiO2 NP sizes (25, 45, and 85 nm in diameter) and at different pH was followed by quartz crystal microgravimetry (QCM). Film buildup, as assessed by QCM mass changes, was 3 times larger when assembled with PAH at pH 9 than with PAH at pH 5. After chemically cross-linking the PAA/PAH within the films, the SiO2 NPs were removed by dissolution with hydrofluoric acid. Atomic force microscopy and transmission electron microscopy were used to characterize the porous films formed and revealed that the pore size could be controlled by using SiO2 NPs of different size. The adsorption of bovine serum albumin (BSA) on the porous thin films showed that the amount of BSA adsorbed increased with increasing bilayer number, suggesting the protein infiltrated the films. This provides opportunities to tune the protein content in the films, which is of interest for applications in biocatalysis and biosensing.