Enzymes are used to amplify signals for detection of antigen proteins in biological samples. However, the enzymes conventionally used for this purpose have limitations, such as the presence of the same (i.e., endogenous) activity in human cells and difficulty in simultaneous use of multiple enzymes because of differences in their required reaction conditions. In this report, we identify an enzyme that can overcome these problems: β-D-galacturonidase (GalUAase) from Eisenbergiella tayi. GalUAase activity was confirmed to be absent from human cells. The substrate of GalUAase, galacturonic acid, is highly hydrophilic because of its anionic carboxylate group; high substrate hydrophilicity is an ideal characteristic for the substrate of an enzyme used for detection because it decreases nonspecific adsorption to biological samples. We show that E. tayi GalUAase could be used in the detection of antigen proteins on live human cells with lower background signal than the conventionally used enzyme β-D-galactosidase. The combinatorial use of GalUAase with β-D-galactosidase enabled simultaneous detection of two antigens on live cells.