• A MnO 2 shell-isolated SERS nanoprobe (Au-Mpy-Au-MnO 2 ) is prepared for quantitative detection of ALP activity. • Due to the efficient physical isolation of MnO 2 shell, the nanoprobe demonstrates high stability against salt and thiol. • The sensing performance is achieved by enzyme-triggered etching of MnO 2 shell. • With MnO 2 shell as Raman reference, this system effectively eliminates the experimental interferences. • Integrating with in-capillary technique, this SERS system is capable of direct quantitative detection in trace serum. Establishing a stable, reliable and sensitive assay for the direct analysis in trace serum is of significance for the medical diagnosis and disease monitoring. We develop a MnO 2 shell-isolated SERS nanoprobe (Au-Mpy-Au-MnO 2 , AMAM) for the quantitative detection of alkaline phosphatase (ALP) in trace serum. Due to the physical isolation of MnO 2 shell, AMAM is equipped with high stability, rarely happening the accidental aggregation. Particularly, the SERS signal is regulated by the etching of MnO 2 shell, requiring not to change the local electromagnetic field or the molecular structure of Raman reporter (4-mercaptopyridine (Mpy)). Designating MnO 2 shell as the Raman reference and the ratio of I Mpy /I MnO2 as the response signal, AMAM system can obviously eliminate the experimental interference and achieve highly reliable SERS quantification for ALP. This ratiometric SERS system exhibits good reproducibility, high stability and sensitivity for the ALP analysis. AMAM system is able to perform the colorimetric and SERS dual-readout detection, which displays a wide linear dynamic range (0.1–70 U/L) for ALP with a detection limit of 0.079 U/L. Additionally, integrating with in-capillary SERS technique, our proposed nanoprobe is also applicable of direct SERS analysis in trace undiluted human serum (2 μL).