Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, represent an emerging paradigm in precision diagnostics. Despite the utility of DNA barcodes as a multiplexing tool, their susceptibility to nucleases in vivo has limited their utility. Here we exploit chemically stabilized nucleic acids to multiplex synthetic biomarkers and produce diagnostic signals in biofluids that can be ‘read out’ via CRISPR nucleases. The strategy relies on microenvironmental endopeptidase to trigger the release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed urine. Our data suggest that DNA-encoded nanosensors can non-invasively detect and differentiate disease states in transplanted and autochthonous murine cancer models. We also demonstrate that CRISPR-Cas amplification can be harnessed to convert the readout to a point-of-care paper diagnostic tool. Finally, we employ a microfluidic platform for densely multiplexed, CRISPR-mediated DNA barcode readout that can potentially evaluate complex human diseases rapidly and guide therapeutic decisions.