Nucleic acids are well-established biomarkers of cancer with immense value in diagnostics and basic research. However, strategies to monitor these species in tissue can be challenging due to the need for amplification of imaging signal from low analyte concentrations with high specificity. Photoacoustic (PA) imaging is gaining traction for molecular imaging of proteins, small biomolecules, and nucleic acids by coupling pulsed near-infrared (NIR) excitation with broadband acoustic detection. This work introduces a PA nucleic acid contrast agent that harnesses NIR fluorophore and quencher-tagged hybridization chain reaction (HCR) for signal amplification. This HCR probe was designed to enable contact quenching between NIR dye-quencher pairs by coercing their direct alignment when miR-21, a microRNA cancer biomarker, is detected. The probe demonstrated a ratiometric PA limit of detection of 148 pM miR-21, sequence specificity against one- and two-base mutations, and selectivity over other microRNAs. It was further tested in live human ovarian cancer (SKOV3) and noncancerous (HEK 293T) cells to exemplify PA activation based on differences in endogenous miR-21 regulation ( = 0.0002). The probe was lastly tested in tissue mimicking phantoms to exemplify sustained contrast in centimeter-range depths and 85.3% photostability after 15 min of laser irradiation. The probe’s miR-21-specific activation and its ability to maintain contrast in biologically relevant absorbing and scattering media support its consideration for live-cell PA microscopy and potential cancer diagnostics. Results from this probe also underscore the combined detection power between ratiometric PA signaling and strand amplification for more sensitive DNA-based PA sensors.