Accurate staging by sentinel lymph node (SLN) biopsy is essential for improving prognostic outcomes of canine mammary tumors (CMT). Indocyanine green near-infrared fluorescence (ICG-NIRF) imaging offers a novel real-time approach for SLN mapping, potentially enhancing the precision of surgical SLN biopsies. This study evaluated the effectiveness of transcutaneous ICG-NIRF imaging in identifying lymphatic drainage pathways and SLNs in CMT and determined the optimal ICG concentration for visualizing lymphatic channels and SLNs. We hypothesized that transcutaneous ICG-NIRF imaging effectively highlights both lymphatic channels and SLNs, facilitating accurate SLN biopsies. This study included 24 female dogs with cytologically diagnosed malignant CMT, from September 2023 to April 2024. ICG was injected peritumorally (concentrations: 0.5, 1.0, and 2.5 mg/mL). NIRF imaging was conducted intraoperatively to visualize lymphatic drainage and identify SLNs. Data on signal-to-background ratio of lymphatic drainage and SLN and operation time were collected and analyzed using ANOVA and post-hoc Tukey tests. The detection rate of SLNs using ICG-NIRF was 94.4% (34/36). Highest LN fluorescence (p = 0.030) and shortest operation time (p = 0.002) were yielded by 1.0 mg/mL ICG, with minimal false-negative rates (0%, 0/18). ICG-NIRF imaging enhances the accuracy of sentinel lymph node mapping in CMT by enabling real-time visualization of lymphatic drainage, optimizing lymph node biopsy selection, and improving surgical precision, thereby contributing to more accurate tumor staging and better prognostic assessment.