Current strategies for sentinel lymph node (SLN) biopsy to detect cancer metastasis have some limitations such as the associated radiation exposure and high false-negative rates due to rapid diffusion of the dye particles through the true SLNs to contiguous lymph nodes. In this study, we incorporated the clinically used indocyanine green (ICG) into biocompatible poly (lactic-co-glycolic acid) (PLGA) microbubbles, and filled these with perfluorocarbon gas to form near-infrared (NIR) fluorescent polymer (ICG-PLGA) microbubbles for more efficient sentinel lymph node imaging. With the encapsulation of ICG into PLGA, the photo-stability of aqueous ICG solution was markedly enhanced at both room- and body-temperature, and could be concurrently used as a contrast media to enhance near-infrared fluorescence (NIRF) imaging and ultrasound (US) imaging signals. Upon subcutaneous injection into the front paw of a mouse, ICG-PLGA microbubbles translocated via lymphatic vessels into SLNs within 5 min; the associated NIR fluorescent signal intensity and retention time in lymph node was superior to that achieved with use of ICG alone. Further, ICG-PLGA microbubbles enhanced ultrasonographic contrast of popliteal lymph nodes of rabbits, unlike a simple ICG solution. Use of ICG-PLGA microbubbles improved efficacy of SLN imaging in this study; their use as imaging contrast agents for SLN mapping in humans is expected to be of much clinical relevance.