Breast-conserving surgery (BCS) is facing the challenge of objective tumor margin identification intraoperatively. Near-infrared fluorescence imaging (NIR-FI) would be an ideal approach to visualize tumor margins during surgeries. In this preliminary study, the feasibility of methylene blue (MB) based NIR-FI technique for breast cancer detection were assessed in resected human breast specimens after breast cancer surgeries. Thirty breast cancer patients scheduled for surgical treatment were enrolled, including 10 patients with preoperative chemotherapy and 20 patients without. Each of them received an injection of 1 mg/kg MB intravenously 3 hours before the surgery. Then, a home-developed MB specific NIR-FI system was employed to imaging the resected breast tissues and identify the tumor by the fluorescence contrast. Specimens were taken for pathological examinations as the reference. There were no severe adverse events attributable to MB. 16/20 patients, who did not receive preoperative chemotherapy, exhibited fluorescent contrast on their resected tissues (SBR: 1.94 ± 0.71). In contrast, tumors were identified in 3/10 specimens from patients who underwent preoperative chemotherapy (SBR: 1.63 ± 0.38). Total 35 tissues were sampled from 30 specimens. Besides 30 tumor samples, 5 more suspicious samples with fluorescence signal were confirmed to be benign hemorrhagic tissues. Therefore, a sensitivity of 0.63 and a positive predictive value of 0.79 were achieved by the MB fluorescence imaging strategy. Here we demonstrate the feasibility of using MB-FI to identify breast cancer. Preoperative chemotherapy had an impact on imaging effect, which may reduce the detection rate. After all, MB-FI has great potential to be used into BCS for tumor-positive margins detection, but further clinical trial study is needed. (http://www.chictr.org.cn/ Clinical Trial Registry ID: ChiCTR1800015400, Near-infrared fluorescence imaging applied in breast cancer identification with Methylene Blue)