Enzyme activity is the basis for many biosensors where a catalytic event is used to detect the presence and amount of a biomolecule of interest. To create a practical point-of-care biosensor, these enzymes need to be removed from their native cellular environments and immobilized on an abiological surface to rapidly transduce a biochemical signal into an interpretable readout. This immobilization often leads to loss of activity due to unfolded, aggregated, or improperly oriented enzymes when compared to the native state. In this work, we characterize the formation and surface packing density of a stable monolayer of acetylcholinesterase (AChE) immobilized on a planar gold surface and quantify the extent of activity loss following immobilization. Using spectroscopic ellipsometry, we determined that the surface concentration of AChE on a saturated Au surface in a buffered solution was 2.77 ± 0.21 pmol cm