Sentinel lymph node (SLN) biopsy is a reliable predictor of lymph node status in staging solid cancers and it is generating a great deal of enthusiasm among clinicians. Herein, methylene blue (MB) was encapsulated within a microemulsion nanosystem. Its near-infrared (NIR) fluorescence was used for mouse SLN imaging and mapping. The results show an obvious improvement in the NIR fluorescence of the MB aqueous solution after the MB was encapsulated into the microemulsion. When a mouse was intradermally injected with the microemulsion-encapsulated MB (ME-MB) into the paw, its axillary SLN rapidly emitted brighter fluorescence than that of the SLN of a mouse intradermally injected with the MB aqueous solution only. Furthermore, the retention time of the fluorescent signal emitted from the SLN mapped with the ME-MB was longer than that of the fluorescent signal emitted from the SLN mapped with the MB aqueous solution only. This was due to the microemulsion being a lymphatic targeting nanosystem, and the ME-MB having brighter fluorescence, thereby giving the operator enough time to identify and ensure complete resection of the SLN. SLN mapping with the NIR fluorescence of ME-MB nanosystem is a simple, intuitive and highly efficient technique compared to traditional methods.