A dual-mode imaging technique has been developed for intraoperative imaging of bile ducts and real-time identification of iatrogenic injuries in cholecystectomy. The technique is based on ultrasound (US) and fluorescence (FL) imaging of a dual-mode microbubble (MB) agent comprising a poly (lactic-co-glycolic acid) (PLGA) shell and a core of Indocyanine Green. During cholecystectomy, a clinical US probe is used to localize the bile duct structure after bolus injection of dual-mode MBs. As the surrounding adipose tissue is removed and the Calot's triangle is exposed, FL imaging is used to identify the MB distribution and to determine the potential bile duct injury. The contrast-enhanced bile duct imaging technique has been demonstrated in both a surgical simulation model and an ex vivo porcine tissue model under two surgical scenarios. The first scenario simulates the correct procedure where the cystic duct is clipped. The second scenario simulates the incorrect procedure where the common bile duct is clipped, leading to consequent bile duct injury. Benchtop experiments in both the phantom and the ex vivo models show that the dual-mode imaging technique is able to identify the potential bile duct injury during cholecystectomy. A phantom system has also been established for future device calibration and surgical training in image-guided cholecystectomy. Further in vivo animal validation tests are necessary before the technique can be implemented in a clinical setting.