In this study, we developed ratiometric surface-enhanced Raman scattering (SERS) biosensors using Ag-Au alloy nanoflowers as SERS substrates, molecules having amide bonds and alkyne groups (Tag A) as Raman reporters, and sodium thiocyanate as an internal standard molecule (Tag B) for the sensitive detection of human carboxylesterase-1 (hCE1) in HepG-2 cells. The correlation between HepG-2 cell damage and hCE1 activity levels was investigated. Both Tag A's alkyne group and Tag B's cyanide group produced characteristic SERS signals in the Raman-silent region (I_{2000 cm^-1} and I_{2115 cm^-1}, respectively). The hydrolysis of the amide bond in Tag A via hCE1 and the shedding of the alkyne group led to a reduction in the SERS signal intensity observed at I_{2000 cm^-1}. Conversely, the SERS signal intensity of Tag B at I_{2115 cm^-1} exhibited a consistent pattern. As the activity level of hCE1 and the ratiometric peak intensity (I_{2000 cm^-1}/I_{2115 cm^-1}) correlated negatively, hCE1 could be quantitatively detected within the range of 10^-2 to 2 × 10² ng·mL^-1, with a detection limit of 7.3 pg·mL^-1. The ratiometric SERS probe strategy, in which a ratio response is employed, permits sensitive and reproducible SERS detection by facilitating intrinsic calibration to rectify signal fluctuations resulting from temporal and spatial variations in the detection conditions. Concurrently, the implementation of Raman-silent region reporter molecules mitigates the interference from endogenous biomolecules in SERS measurements and offers a novel approach for achieving highly sensitive and interference-free detection of intracellular hCE1.

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