Abstract Porous films were fabricated from nonporous layer-by-layer multilayers composed of a blend of positively charged disulfide-containing polyamidoamine and poly(allylamine hydrochoride), and negatively charged poly(acrylic acid), followed by removal of cleavable disulfide-containing polycation after incubation in 1 mM DTT solution. The thickness of original multilayered films decreased with the increase of incubation time in DTT solution. Atomic force microscopy (AFM) measurements and electrochemical analysis demonstrated the formation of nanopores with sizes ranging from 50 to 120 nm. The formed porous films were stable in buffer solution at pHs ranging from 7.4 to 1.6, whereas they showed slight changes in pore number and pore size when incubated in PBS buffer at a pH of 10.0. This research might provide a universal method for the fabrication of noncrosslinked porous multilayered films.