The noncovalent immobilization of alkaline phosphatase (ALP) onto aqueous dispersed nylon 6 nanorods (310 nm mean diameter; 6 μm mean length) prepared by anodic aluminum oxide (AAO) membrane templating was studied. Using multi-stacked layer-by-layer (LBL) assembly with the cationic quaternary ammonium polymer Sapphire II™, the amount of alkaline phosphatase (ALP) enzyme loaded onto the polymer nanostructures was found to be 115 ± 7 μg mg−1 nanorod. The biofunctionalized nanorods were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD™. The results indicate that the kinetic parameters, Km and Vmax, for the catalytic activity of the nanostructure-bound alkaline phosphatase (ALP) enzyme are different from those of soluble (‘free’) ALP. While the Km value was measured to be 156 μM for free ALP, the apparent Km value determined for the LBL-immobilized ALP is approximately 20% lower (122 μM). Furthermore, despite the relatively high enzyme loading capacity of the nanorods, the specific activity of the bound ALP enzyme was found to be almost nine times lower than that measured for free ALP. Finally, additional experiments revealed that the catalytic activities of both free ALP and nanorod-conjugated ALP are affected similarly by changes in pH, with optimal performance levels occurring under conditions of pH 9.5. To the best of our knowledge, this study represents the first report examining the preparation of aqueous dispersed, AAO-templated polymer nanorods for potential application as enzyme scaffolds in chemiluminescent-based assay systems.