Propranolol (PPL) imprinted microspheres (MIP) were successfully prepared via oil/water polymerization using a methyl methacrylate (MMA) monomer, PLL template, and divinylbenzene (DVB) cross-linker and favorably incorporated in a Eudragit-RS100 nanofiber membrane. A non-PPL imprinted polymer (NIP), without a template, was used as a control. The morphology and particle size of the beads were investigated using scanning electron microscopy. The results revealed that both MIP and NIP had a spherical shape with a micron size of approximately 50-100 mu m depending on the amounts of DVB and PPL used. NIP2 (MMA/DVB, 75: 2.5) and MIP8 (PPL/MMA/DVB, 0.8: 75: 2.5) were selected for reloading of PPL, and the result indicated that increasing the ratio of PPL to polymer beads resulted in increase PPL reloading (>80%). A total of 10-50% NIP2 or MIP8 was incorporated into a 40%(w/v) Eudragit-RS100 fiber membrane using an electrospinning technique. PPL could be bound to the 50% MIP8 composite fiber membrane with a higher extent and at a higher rate than the control (NIP2). Furthermore, the MIP8 composite fiber membrane showed higher selectivity to PPL than the other beta-blockers (atenolol, metoprolol, and timolol). Thus, the MIP8 composite fiber membrane can be further developed for various applications in pharmaceutical and other affinity separation fields.