Porous nanostructures of polypyrrole (Ppy) were fabricated using colloidal lithography and electrochemical techniques for potential applications in drug delivery. A sequential fabrication method was developed and optimized to maximize the coverage of the Ppy nanostructures and to obtain a homogeneous layer over the substrate. This was realized by masking with electrophoretically-assembled polystyrene (PS) nanospheres and then electroplating. Drug/biomolecule adsorption and the release characteristics for the porous nanostructures of Ppy were investigated using rhodamine B (Rh-B). Rh-B is an easily detectable small hydrophobic molecule that is used as a model for many drugs or biological substances. The porous Ppy nanostructures with an enhanced surface area exhibited higher Rh-B loading capacity than bulk planar films of Ppy. Moreover, tunability of surface morphology for further applications (e.g., sensing, cell adhesion) was demonstrated.