Display omitted •Natural dyes were in situ identified using SERS method on fibers.•Ag nanoparticles SERS substrate was prepared by laser photoreduction.•Ag dendrites SERS substrate was prepared by galvanic displacement.•SERS substrates had characteristics of high sensitivity and reproducibility.•Different dye species were distinguished in one archaeological fiber. Accurate detection of natural dyes in archaeological textiles is challenging due to limited sample availability. In situ detection of fibers by surface-enhanced Raman scattering (SERS) can overcome this challenge. In this study, we present two methods for the preparation of SERS substrates based on the laser photoreduction reaction and the galvanic displacement reaction, respectively. Our results show that the substrates prepared by these methods, including 450 nm-AgNPs, Cu-based Ag, and Zn-based Ag, offer several advantages such as rapid preparation, easy handling, low cost, high sensitivity and excellent reproducibility. Using these substrates, we successfully detected anthraquinones (alizarin, purpurin), indigoids (indigotin, indirubin), and protoberberine-type dyes in archaeological textiles from the Wei and Jin Dynasties. Notably, we were able to detect both indigo and protoberberine-type dyes in one archaeological fiber. The minimally invasive, in situ analytical approach developed provides a valuable tool for analytical applications where sampling is limited to microscopic amounts.