Early detection and diagnosis are the most important endeavors for reducing associated morbidity and mortality of pancreatic ductal adenocarcinoma (PDAC). Developing molecular imaging probes that can specifically and effectively target cancer-associated biological pathways is one of the key points for sensitive and accurate diagnosis for PDAC. Herein, a small-sized, bispecific fusion protein constructed by genetic fusion of different binding domains of antibodies, termed Bi50, with enhanced targeting effect for PDAC is reported. Bi50 has excellent bispecific targeting for vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) simultaneously in vitro and in vivo. Additionally, Bi50 shows increased intratumoral permeability and enrichment characteristics in the tumor than the control protein, which is constructed directly connecting two individual Fabs. Moreover, Bi50 can not only target areas rich in vasculature but also bind with affinity to tumor parenchymal cells, achieving “multilevel” targeting effect. Our work demonstrates that the bispecific fusion protein Bi50 has great potential as an efficient, targeted molecular imaging probe.Copyright © 2020. Published by Elsevier Ltd.