Human orthogonal enzymes (HOEs), do not show the same activities as the endogenous enzymes of human cells and thus are useful as amplification enzymes to detect antigen proteins in biological samples. Here, we evaluates a new HOE from Escherichia coli, α-sulfoquinovosidase (α-SQase). We confirmed that the activity of α-SQase did not exist in examined human cell lines, and thus it was applicable to live-cell enzyme-linked immunosorbent assay (ELISA) in which the antigen membrane protein on cells was detected without inactivating endogenous enzymes, a pretreatment required for cell ELISA using conventional amplification enzymes. Here, we also developed a fluorescent substrate for α-SQase whose active residue is located at the end of the narrow, deep pocket of the substrate recognition site. The designed methylumbelliferyl substrate with a hydroxyl benzyl alcohol linker showed similar reactivity as the p-nitrophenol substrate, a good substrate for α-SQase.