This study investigated the effect of fiberoptic ductoscopy (FDS) combined with methylene blue staining immediately after FDS procedure on pathological nipple discharge diseases. A retrospective analysis of the clinical data of 122 patients with nipple discharge, who underwent FDS and surgical treatment at the Department of Breast and Thyroid Surgery of Tengzhou Central People’s Hospital, was conducted. The demographic characteristics and surgical outcomes of all patients were assessed. According to the injection time of methylene blue, the patients were divided into the control and the observational groups. In the observational group, methylene blue was injected immediately after ductoscopy and then surgical treatment was performed 12–24 h later, while in the control group, methylene blue injection was just few minutes before surgery treatment. There was no significant difference in the demographic characteristics between the two groups such as age and disease course, in the observational group, the incision length 2.39 (0.48) cm, the volume of resected tissue 41.93 (40.57) cm3, the intraoperative blood loss 12.19 (2.10) ml and the operation duration 26.95 (4.51) min were significantly lower than those of the traditional group (P < 0.05). The average hospital stay 3.08 (0.62) days, breast shape satisfaction 4.78 (1.63) points and postoperative drainage tube placement [3 (5.08%) days] in the observational group were significantly better than those in the control group (P < 0.05). FDS combined with immediate methylene blue staining, which has the advantages of accurate location of the diseased duct, small surgical incision, less tissue removal, and ease of finding the orifice of discharged mammary duct, and is worthy of widespread clinical application.