We propose a portable fluorescence microscopic imaging system (PFMS) for intraoperative display of biliary structure and prevention of iatrogenic injuries during cholecystectomy. The system consists of a light source module, a camera module, and a Raspberry Pi computer with an LCD. Indocyanine green (ICG) is used as a fluorescent contrast agent for experimental validation of the system. Fluorescence intensities of the ICG aqueous solution at different concentration levels are acquired by our PFMS and compared with those of a commercial Xenogen IVIS system. We study the fluorescence detection depth by superposing different thicknesses of chicken breast on an ICG-loaded agar phantom. We verify the technical feasibility for identifying potential iatrogenic injury in cholecystectomy using a rat model in vivo. The proposed PFMS system is portable, inexpensive, and suitable for deployment in resource-limited settings.