Objective To prepare a pancreatic cancer-targeted nano-scale ultrasound contrast agent (T-UCA) and to evaluate its in vitro targeting effect. Methods PLGA-PEG-NHS was synthesized with poly(lactic-co-glycolic acid) (PLGA), N-hydroxysuccinimide (NHS) and polyethylene glycol (PEG). The construction of PLGA-PEG-NHS was characterized by ¹H-NMR. Perfluoroctyl bromide (PFOB)-loaded PLGA nanoparticle contrast agent was prepared using emulsion evaporation technique with PLGA-PEG-NHS and PFOB, and the products were further conjugated with Hedgehog antibody. The morphology of T-UCA were characterized by transmission electron microscopy, and the size distribution and Zeta potential of T-UCA were characterized by dynamic light scattering method. Furthermore, the drug entrapment efficiency and loading capacity of T-UCA were determined by GC-MS,and the release rate of T-UCA in vitro was examined by dialysis method. Finally, the in vitro targeting performance was quantitatively verified by fluorescence microscopy and flow cytometry with human pancreatic cancer lines SW1990 and CFPAC-1. Results The average diameter and the Zeta potential of T-UCA were 198.9 nm and -31.8 mV, respectively. Moreover, the encapsulation efficiency and drug loading of T-UCA was (63.7 ± 3.9)% and (14.3 ± 0.9)%, respectively. Nearly 85.3% liquid perfluorocarbon was released from the T-UCA within 48 h. In vitro cell experiments showed that the targeted contrast agent could bind to SW1990 cells which had high expression of Hedgehog antigen, while not to the CFPAC-1 cells without expression of Hedgehog antigen. Conclusion The emulsion evaporation technique can be used to prepare T-UCA with desirable characteristics, and the prepared T-UCA can specifically target the pancreatic cancer cells with high expression of Hedgehog, making it a promising pancreatic cancer-targeted nanosacle ultrasound contrast agent.