Aquaporin, a transmembrane protein that functions as a highly selective water channel, has intrigued membrane researchers around the world who have attempted to synthesize membranes embedded with aquaporin for water purification. We have molecularly designed pore-suspending biomimetic membranes embedded with Aquaporin Z (AQPz) based on different lipid protein ratios and investigated their local mechanical stabilities by force indentation using atomic force microscopy (AFM). The compatibility between 1,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC) and AQPz was proved by stop-flow experiments, in which the DMPC-AQPz (a lipid to protein molar ratio of 2000:1) exhibited 3000-fold higher permeability than the pristine DMPC vesicles. A series of vesicles were then ruptured on pristine porous alumina and carboxylated polyethylene glycol (PEG) coated porous alumina. The carboxylated-PEG polymer cushion significantly enhances the flexibility of the DMPC bilayer on the substrate and also gives rise to higher breakthrough forces from AFM when AQPz is reconstituted into the pore-suspending membrane on the carboxylated-PEG coated substrate. The local membrane mechanical stability is further improved by increasing the AQPz content, suggesting that AQPz increases the energy barrier required for a normal force to punch through the resultant biomimetic membrane. Therefore, the carboxylated-PEG grafted porous alumina exhibits a promising platform for the fabrication of pore-suspending biomimetic membranes. Nevertheless, to achieve the highest performance of DMPC-AQPz biomimetic membranes, a compromise may be needed to optimize the DMPC-AQPz ratio.