Surface-enhanced Raman scattering (SERS) still faces a big challenge in bioanalysis due to the biological background interference and the poor reproducibility of the Raman signal. Accordingly, herein a novel surface-enhanced Raman scattering (SERS) nanoprobe is prepared via the co-assembly of 3-(4-(phenylethynyl)benzylthio) propanoic acid (PEB) tagged-peptides and 4-thiol phenylacetylene (TPA) on gold nanorods, and used for the measurement of caspase 3, as a biomarker. A key advantage of this system is the lack of background alkyne signals throughout the cell. The two alkyne-bearing molecules produce distinct SERS signal but the PEB signal decreases in response to the peptide-cleavage activity of caspase 3, thereby allowing the ratiometric detection of analytes. Under the optimized conditions, the ratiometric peak intensity of I2027/I2218 dynamically increased with increasing caspase 3 concentration in the range from 12.5 to 500 ng/mL, with a detection limit of 1.99 ng/mL based on a signal-to-noise ratio of S/N = 3. The nanoprobe has been successfully applied in a live cell imaging assay of caspase 3 and in an ischemia-reperfusion surgery-treated rat living tissue model.,Copyright © 2018 Elsevier B.V. All rights reserved.