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The precise detection of pathological type I collagen is critical for diagnosing and prognosticating various diseases such as fibrosis and tumors. Surface-enhanced Raman scattering (SERS) is emerging as a promising high-resolution imaging technique, yet the intricate process of covalently modifying antibodies often results in SERS probes with diminished selectivity. We herein for the first time report the development of dual SERS peptide probes, Ag@R1@PCTP (S-P) and Ag@R2@ICTP (S-I), which integrate collagen targeting peptides (CTPs), Raman reporters, and silver nanoparticles (Ag NPs) for specific imaging of pathological type I collagen in connective tissues. The S-P/S-I dual SERS peptide probes displayed distinct peaks at 2154 cm−1 and 2227 cm−1 in the Raman silent region with no overlap, showcasing their capability for multiplex imaging free from cross-interference. The SERS peptide probe S-P is specifically tuned to target pathological collagen, excluding intact collagen, while S-I effectively recognizes type I collagen in both its pathological and healthy states. The S-P/S-I probes have exhibited outstanding effectiveness in identifying pathological type I collagen across various connective tissues and have been successfully applied for imaging this collagen in a variety of cancer tissues. The innovative dual SERS peptide probes offer a highly specific and robust tool for imaging pathological type I collagen in tissues, presenting significant potential for the accurate diagnosis and treatment of a range of collagen-related diseases. • A highly specific sensor for SERS imaging of pathological type I collagen was established. • SERS peptide probes S-P and S-I showed selective identification of pathological collagen and type I collagen, respectively. • Dual SERS peptide probes have been effectively deployed for targeting pathological type I collagen in diverse tissues. [ABSTRACT FROM AUTHOR]