We investigated localization and safe resection margins for breast cancer patients undergoing breast conserving surgery (BCS) using ultrasound-guided indocyanine green fluorescence (ICG-F) marking. From April 2016 to March 2019, we prospectively enrolled 114 patients who underwent BCS using US-guided ICG-F marking and we compared these results with 300 patients who underwent BCS using US-guided skin marking from January 2012 to December 2016. Clinical features, identification rates, status of resection margins, and re-operation rates were analyzed. The ICG-F identification rate was 100% (114/114). The mean approach time for resection of the lesion ICG-F using group was about 13 min. The positive rate of frozen resection margins was 10.5% using ICG-F and 25.0% using sono-guided skin marking (p < 0.01). The rate of additional intraoperative resection was significantly lower in the ICG-F marking group compared to that in the sono-guided skin marking group (8.8% vs. 23.3%, p < 0.01). The rate of final positive resection margins was 3.5% in the ICG-F using group and 14.7% in the sono-guided skin marking group (p < 0.01). The rate of re-operation was 4.4% in the ICG-F using group and 4% in the sono-guided group (p = 0.79). At follow-up after the operation using ICG-F, no complications occurred. Using ICG-F during BCS could be a safe, sophisticated method for localization.