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A novel surface-enhanced Raman scattering and lateral flow assay (SERS–LFA) biosensor with multiple channels based on an aptamer has been proposed in this work. The developed biosensor was designed for the rapid, convenient, sensitive and specific detection of vascular endothelial growth factor (VEGF) and osteopontin (OPN) in several samples simultaneously. During the assay, VEGF and OPN were recognized by detection aptamers immobilized on cuprous oxide nanocubes (Cu2O NCs) and captured by capture aptamers to form a sandwich structure on the test lines. In this way, generous numbers of "hot spots" formed by the aggregation of Cu2O NCs remarkably enhanced the SERS signals of Raman reporters (NBA and 4-ATP). The detection limits were as low as 0.78 pg mL−1 for the VEGF and 0.86 pg mL−1 for OPN in the human serum within 15 min, respectively. Additionally, the biosensor possesses favorable specificity, reproducibility, uniformity and stability. In the clinical analysis of serum specimens, patients with cervical cancer can be differentiated from healthy volunteers. Consequently, the presented aptamer-based SERS–LFA biosensor with multiple channels provides a powerful tool for the early diagnosis of cervical cancer. [ABSTRACT FROM AUTHOR]