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Cervical cancer is a common cause of cancer-related death in women worldwide, and quantitative detection of serum tumor markers has practical implications for diagnosing cervical cancer. In this study, a novel surface-enhanced Raman scattering (SERS)-based immunosensor for the hypersensitive simultaneous detection of survivin and osteopontin (OPN) in cervical cancer serum was proposed, which was composed of the AuNPs@SiO2 microspheres (Au@SiO2) array immunosubstrate and Au-Ag nanoshells (Au-AgNSs) immunoprobes. The experimental results demonstrated that the constructed SERS immunosensor exhibited satisfactory selectivity and reproducibility while possessing detection limits as low as 0.908 pg/mL and 0.813 pg/mL for survivin and OPN in human serum. Finally, its usefulness in real serum samples (from healthy subjects, cervical intraepithelial neoplasia 1 (CIN 1), CIN 2, CIN 3 and cervical cancer patients) was verified, and the detection data were in good agreement with the enzyme-linked immunosorbent assay results. This immunosensor has excellent performance, which is promising for the early screening of cancer. The ordered single-layer gold film and monolayer SiO2 array were obtained by the oil–water interface self-assembly method and Langmuir-Blodgett (LB) gas–liquid interface assembly method, respectively. Then, these two ordered structures are combined to prepare Au@SiO2 array substrates. This ordered array substrate not only ensures good uniformity, but also improves SERS performance. [ABSTRACT FROM AUTHOR]