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The characterization of ancient silk provides significant historical information and contribute to the identification of the origin of the material. At present, there are many ways to characterize silk. However, on-site identification of ancient silk remains a crucial challenge. In this study, a paper chromatography system consisting of a colloidal gold immunochromatographic strip (GICS) and a time-resolved fluorescence immunochromatographic strip (TICS) is proposed to meet this challenge. On the basis of the original paper chromatography technique, a monoclonal antibody against silk fibroin (SF) was prepared and applied in this new paper chromatography system. Both the GICS and the TICS showed good sensitivity, specificity and reliability, with the limit of detection for silk fibroin being 30 μg/mL and 49.20 ng/mL, respectively. Moreover, there was no cross reaction with possible interferents. Furthermore, the GICS had acceptable thermal stability. The recovery of the TICS assay was in the range from 98.12% to 103%, and the variation coefficient was from 5.34% to 6.93%. This work significantly improves access to scientific information at archaeological sites, providing new scientific evidence for exploring the origin and transmission roadmap of ancient silk. [ABSTRACT FROM AUTHOR]