The purpose of this study was to evaluate the expression of ser-miRNAs at different periods during treatment and analyze their relationship with therapeutic response and prognosis in HER2-positive breast cancer patients receiving neoadjuvant chemotherapy combined with trastuzumab (NCCT).Venous blood was drawn from patients at different periods during NCCT. The expression of ser-miRNAs was assessed by qRT-PCR and their relation to treatment response and survival was analyzed.The results showed the expression of miR-10b, -21, -34a, -125b, -145, -155, and -373 in patients before the start of treatment was significantly higher, ser-miR-210 was lower, and ser-miR-122 was comparable to the levels in healthy controls. Changes in ser-miR-21 levels during NCCT were significantly correlated to clinical response and survival and, however, were not associated with pathology response. The expression levels of ser-miR-21 were decreased from the start of NCCT to the end of the second cycle and from the start to the end of NCCT in clinical responders; however, there was no significant difference in non-responders. The patients with decreased ser-miR-21 expression from the start to the end of the second cycle and from the start to the end of NCCT had better overall survival (OS) and disease-free survival (DFS) than those with elevated ser-miR-21 expression.These results showed that changes in ser-miR-21 levels were significantly related to NCCT clinical response and prognosis. Ser-miR-21 may serve as a non-invasive biomarker to predict NCCT response in HER2-positive breast cancer.