Beginning with the discovery of the middle Cambrian Burgess Shale, the rare examples of exceptional preservation of soft tissues in fine-grained strata have been celebrated as windows into the diversity and complexity of ancient marine animal life. Even so, with the loss of original organic compounds, the physiological attributes of the fossils can be inferred only on the basis of modern descendants or sister groups among extant representatives that can serve as analogs. To test whether soft-bodied fossils from the Burgess Shale and similar Cambrian Lagerstätten could preserve chemical evidence of their original biology, a number of specimens from different groups were analyzed with synchrotron-based X-ray fluorescence imaging to determine the spatial distribution of a range of elements, especially those in exceedingly trace amounts that cannot be detected by more conventional analytical methods. The relative distribution of common elements like Fe, K, Ti, and Ca is related to the composition of the host shale, abundance of pyrite, and presence or absence of preserved exoskeletal calcite. However, the black stain commonly preserved with the arthropod Marrella splendens is preferentially enriched in Cu due to localized residue in the carbon and minute crystals of chalcopyrite. This is interpreted as indicative of the original chemical composition of a primary biotic fluid, likely blood. Since modern-day arthropods generally utilize Cu-containing hemocyanin for oxygen transport, it is hypothesized that hemocyanin was the biogenic source of the observed Cu. This is the first chemical evidence for blood composition in the invertebrate fossil record. These observations reinforce the consensus molecular clock age for the hemocyanin gene family, which traces its origins back to the ‘Cambrian explosion’, thus providing evidence for the early origin of this oxygen carrier among some arthropod groups.