Purpose <p>Pathological nipple discharge (PND) is associated with malignancy. This study aimed to investigate the value of fiberoptic ductoscopy (FDS) and the feasibility of immediate injection of methylene blue after FDS to identify discharging ducts and intraductal lesions without overflow of methylene blue during surgery.</p> Methods <p>From May 2019 to December 2023, 164 PND patients were enrolled. Methylene blue was injected into the discharging ducts immediately after FDS. Surgery was underwent several hours later. The dyeing effect and the operation time were assessed. The pathological results were analyzed with clinical characteristics and ductoscopic appearances.</p> Results <p>The overall detection rate of malignancy was 14.0% (23/164). Both ultrasound (US) and mammography (MG) were negative in 80 (48.8%) patients, while pathology yielded 10 (12.5%) breast cancers. Statistical analysis revealed that patients exhibiting older age, menopause, positive MG, and bloody discharge had a higher propensity for malignancy (<i>P</i> &lt; 0.05). Ductoscopic features such as multiple and distal lesions, irregular morphology and hemorrhage of the lesions, and roughness and stiffness of the ductal walls were associated with malignancy (<i>P</i> &lt; 0.05). Conducting surgery 12–24&#xa0;h after injection of methylene blue resulted in optimal dyeing without overflow of methylene blue.</p> Conclusion <p>FDS is an effective and feasible examination for PND patients with negative imaging results. Immediate injection of methylene blue after FDS allows clear identification of discharging ducts and intraductal lesions without overflow of methylene blue. This approach may be useful in guiding selective ductectomy for the detection of early breast cancer.</p>