Medieval manuscripts, carefully curated and conserved, represent not only an irreplaceable documentary record but also a remarkable reservoir of biological information. Palaeographic and codicological investigation can often locate and date these documents with remarkable precision. The York Gospels (York Minster Ms. Add. 1) is one such codex, one of only a small collection of pre-conquest Gospel books to have survived the Reformation. By extending the non-invasive triboelectric (eraser-based) sampling technique eZooMS, to include the analysis of DNA, we report a cost-effective and simple-to-use biomolecular sampling technique for parchment. We apply this combined methodology to document for the first time a rich palimpsest of biological information contained within the York Gospels, which has accumulated over the 1000-year lifespan of this cherished object that remains an active participant in the life of York Minster. These biological data provide insights into the decisions made in the selection of materials, the construction of the codex and the use history of the object.