Polyelectrolyte (PE) nanogels consisting of cross-linked PE networks integrate the advanced features of both nanogels and PEs. The soft environment and abundant intrinsic charges are of special interest for enzyme immobilization. However, the crucial factors that regulate enzyme encapsulation and activation remain obscure to date. Herein, we synthesized cationic poly (dimethyl aminoethyl methacrylate), PDMAEMA, nanogels with well-defined size and cross-link degrees and fully investigated the effects of different control factors on lipase immobilization. We demonstrate that the cationic PDMAEMA nanogels indeed enable efficient and safe loading of anionic lipase without disturbing their structures. Strong charge interaction achieved by tuning pH and larger particle size are favorable for lipase loading, while the enhanced enzymatic activity demands nanogels with smaller size and a moderate cross-link degree. As such, PDMAEMA nanogels with a hydrodynamic radius of 35 nm and 30% cross-linker fraction display the optimal catalytic efficiency, which is fourfold of that of free lipase. Moreover, the immobilization endows enhanced enzymatic activity in a broad scope of pH, ionic strength, and temperature, demonstrating effective protection and activation of lipase by the designed nanogels. Our study validates the crucial controls of the size and structure of PE nanogels on enzyme encapsulation and activation, and the revealed findings shall be helpful for designing functional PE nanogels and boosting their applications for enzyme immobilization.