Surgical resection is currently the only potentially curative option for patients with pancreatic cancer. However, the 5-year survival rate after resection is only 25%, due in part to high rates of R1 resections, in which cells are left behind at the surgical margin, resulting in disease recurrence. Fluorescence-guided surgery (FGS) has emerged as a method to reduce incomplete resections and improve intraoperative assessment of cancer. Mucin-16 (MUC16), a protein biomarker highly overexpressed in pancreatic cancer, is a potential target for FGS. In this study, we developed a fluorescent MUC16-targeted antibody probe, AR9.6-IRDye800, for image-guided resection of pancreatic cancer. We demonstrated the efficacy of this probe to bind human pancreatic cancer cell lines in vitro and in vivo In an orthotopic xenograft model, AR9.6-IRDye800 exhibited superior fluorescence enhancement of tumors and lower signal in critical background organs in comparison to a nonspecific IgG control. The results of this study suggest that AR9.6-IRDye800 has potential for success as a probe for FGS in pancreatic cancer patients, and MUC16 is a feasible target for intraoperative imaging.