Pancreatic ductal adenocarcinoma (PDAC) has the poorest five-year survival rate of any cancer. Currently, there are no effective diagnostics or chemotherapeutics. Surgical resection is the only curative therapy. However, most patients experience recurrence due largely to challenges in assessing tumor margin status in the operating room. Molecular probes that selectively highlight pancreatic cancer tissue, having the potential to improve PDAC margin assessment intraoperatively, are urgently needed. In this work, a series of red and near-infrared fluorescent probes is reported. Two were found to distribute to normal pancreas following systemic administration. One selectively accumulates in genetically modified mouse models of PDAC, providing cancer-specific fluorescence. In contrast to the small molecule probes reported previously, it possesses inherent affinity for PDAC cells and tissue, and thus does not require conjugation to targeting agents. Moreover, the probe exhibits intracellular accumulation and enables visualization of four levels of structure including the whole organ, tissue, individual cells and subcellular organelles. It can thus promote new strategies for precision image-guided surgery, pancreatic cancer detection, the monitoring of therapeutic outcomes and basic research.