Regards to the global prevalence of diabetes, clinical management should tackle the awkwardness of continuous glucose monitoring systems (CGMS). Although CGMS are commercially accepted, they are still suffering due to their low sustainability and reusability. One method to circumvent these shortcomings is the immobilization of enzymes onto stable carriers. In this contribution, our aim was to build up a highly stable and reproducible enzyme-based host-guest carrier from maltose-modified hyperbranched poly(ethylene imine) (PEI-Mal-C) and polyelectrolyte multilayers (PEMs) to monitor glucose level. Thus, enzymes, such as glucose oxidase (GOx) and horseradish peroxide (POx), were immobilized in core-shell PEI-Mal-C and highly packaged in carrier-based PEM. Herein, the PEM was created using the layer-by-layer protocol, where a consecutive deposition of polyions was achieved. Therein, the polycation PEI-Mal-C was alternatively deposited with different polyanions, e.g., poly(acrylic acid) and heparin (HE), on a solid substrate. The enzyme immobilization, leaching and enzymatic activity were investigated through different modules, including ultraviolet-visible (UV-Vis) spectrophotometer, rheometer, X-ray spectroscopy, contact angle meter, atomic force microscopy and conductometer. To conclude, our approach enabled the use of immobilized GOx/POx for more than one time with the significantly similarly fitted regression calibration curve. It is implied that this work will be the first step to construct a stable hyperbranched glyconanomaterial-immobilized enzyme based on assembled multilayers, with the potential to be applied in a stable and reusable biosensor.