Surface-enhanced Raman scattering (SERS) is an ultrasensitive fingerprint spectroscopy technique. The combined application of SERS and nanozymes has shown excellent prospects in the field of biomedical diagnosis. In the present study, polyaniline@MoS₂@Fe₃O₄@Au (PANI@MoS₂@Fe₃O₄@Au) nanozymes were successfully prepared using hydrothermal reactions. PANI@MoS₂@Fe₃O₄@Au nanozymes have not only dramatically enhanced the peroxidase-like activity but also significantly enhanced the SERS performance. We also studied the effect of different concentrations of HAuCl₄·4H₂O on the SERS activity of nanozymes and found that PANI@MoS₂@Fe₃O₄@Au nanozymes prepared using 2 mg mL⁻¹ HAuCl₄·4H₂O solution exhibited the best SERS performance. We used SERS to study the enzyme kinetics of the nanozymes, and the results showed that the nanozymes have a strong affinity for H₂O₂ and TMB. Finally, we combined PANI@MoS₂@Fe₃O₄@Au nanozymes and glucose oxidase (GOx) to establish a SERS glucose biosensor, which has the advantages of ultrasensitivity, high-precision, and a wide linear response range. The linear response range of glucose is 10⁻¹¹–10⁻³ M, and the detection limit is 10⁻¹² M. More importantly, we have successfully determined the glucose content in human serum samples with satisfactory accuracy. This work provides an essential new strategy for designing composite nanozymes for biocatalysis and provides a new idea for constructing high-sensitivity SERS biosensors.