Chiral D- and L-N-acryloyl aspartic acid (NAsp) polyelectrolyte (PE) surfaces with similar chemical compositions and physical properties but opposite chirality are designed for enzyme immobilization. Enzymes immobilized onto the chiral PE surfaces present high chiral preference, namely L-NAsp PE surface can keep most of the catalytic activity of the immobilized enzymes, however, for enzymes immobilized on D-NAsp PE surface a large decrease in catalytic activity occurred which was 11 times lower compared with L-NAsp PE surface. This phenomenon of chiral effect on enzymes immobilization can be explained by attenuated total reflectance (ATR) and circular dichroism (CD) results. The results exhibited that L-NAsp PE surface could preserve most of the secondary structures of immobilized enzymes while on D-NAsp PE surface with a large conformation alteration. These chiral surface induced differences after enzyme immobilization can be further used for logic operation. These results imply a novel strategy for the design of new enzymes immobilization materials based on the chiral effect and expand the applications of enzymes in biochips, chemical transformations and chiral biodevices.