In this study, streptavidin-functionalized magnetic nanoparticles were developed as a support for enzyme immobilization based on the specific recognition between biotin and streptavidin. The enzyme-immobilizing ability was evaluated using pullulanase as a model enzyme. Magnetic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) nanoparticles were prepared and functionalized with streptavidin using cyanuric chloride. The immobilized enzymes were characterized using UV-vis spectra, Fourier-transform infrared spectroscopy, and transmission electron microscopy. The immobilized pullulanase retained high levels of activity (85.3%) and exhibited significantly improved pH and thermal stability as compared to the free enzyme. At pH 5.5, the relative activity of the immobilized enzyme (75.2%) was significantly greater than that of the free enzyme (15.8%; p < 0.01). After incubation for 360 min at 60 degrees C, the residual activity of the free enzyme was only 21.5%, while the immobilized enzyme retained more than 70.6% of its residual activity. Moreover, the immobilized pullulanase also exhibited excellent recyclability, retaining more than 74.2% of its initial activity after 8 consecutive reuses. These results indicated that streptavidin-coated magnetic nanoparticles have great potential for use as a support for the immobilization of the various enzymes required for continuous biotechnological applications.