Purpose: Sentinel lymph node biopsy (SLNB) has emerged as the preferred standard procedure in patients with breast cancer, melanoma and other types of cancer. Herein, we developed a method to intra-operatively map SLNs and differentiate tumor metastases within SLNs at the same time, with the aim to provide more accurate and real-time intraoperative guidance. Experimental Design: Hyaluronic acid (HA), a ligand of lymphatic vessel endothelial hyaluronan receptor (LYVE)-1, is employed as a SLN mapping agent after being conjugated with a near-infrared fluorophore (Cy5.5). Different sized HAs (5, 10 and 20K) were tested in normal mice and mice with localized inflammation to optimize LN retention time and signal to background ratio. Cetuximab, an antibody against epidermal growth factor receptor (EGFR), and trastuzumab, an antibody against human epidermal growth factor receptor 2 (HER2), were labeled with near-infrared fluorophore (IRDye800) for detecting metastatic tumors. LN metastasis model was developed by hock injection of firefly luciferase engineered human head neck squamous carcinoma cancer UM-SCC-22B cells or human ovarian cancer SKOV-3 cells. The metastases within LNs were confirmed by bioluminescence imaging (BLI). IRDye800-Antibodies were intravenously administered 24 h before local administration of Cy5.5-HA. Optical imaging was then performed to identify nodal metastases. Results: Binding of HA with LYVE-1 was confirmed by ELISA and fluorescence staining. HA with a size of 10K was chosen based on the favorable migration and retention profile. After sequential administration of IRDye800-antibodies intravenously and Cy5.5-HA locally to a mouse model with LN metastases and fluorescence optical imaging, partially metastasized LNs were successfully distinguished from un-metastasized LNs and fully tumor occupied LNs, based on the different signal patterns. Conclusions: Fluorophore conjugated HA is a potential lymphatic mapping agent for SLNB. Dual-tracer imaging with the combination of lymphatic mapping agents and tumor targeting agents can identify tumor metastases within SLNs, thus may provide accurate and real-time intra-operative guidance to spare the time spent waiting for a biopsy result.