Background: The implementation of mammogram-based screening has significantly improved the early detection of breast cancer in the west. However, the use of screening mammography is less prevalent in Asia partly due to social and cultural reasons. The aim of this study was to determine if a serum microRNA (miRNA) panel could be used as biomarkers to assist in the early detection of breast cancer. Methods: We conducted a multi-center, multi-ethnic study to identify and validate miRNA biomarkers for the early detection of breast cancer. A total of 1070 subjects including 550 breast cancer cases (predominantly stage 1 and 2) and 520 matched controls from 6 independent sources were included in this study. Among these, there were 768 American and European subjects recruited by biobanks and 302 Singaporean Asian Subjects recruited at the National Cancer Centre Singapore and the National University Hospital. The study was conducted in 3 phases. First, 119 European Caucasian serum samples (Discovery Cohort) were interrogated to identify differentially expressed miRNAs between early-stage breast cancer cases and matched controls, among 520 circulating miRNA candidates by quantitative RT-PCR using MiRXES assays. The remaining 951 subjects from 5 independent sources were assigned into two groups for biomarker optimization/algorithm development (Optimization Cohort, n = 451) and validation (Validation Cohort, n = 500). Results: Among the 520 circulating miRNAs measured, 241 were quantified in absolute copy numbers for 119 subjects in the Discovery Cohort. Thirty-two candidate miRNAs were identified. These miRNAs consistently showed differential expression between cancer and control subjects in the Optimization Cohort. A multi-variant panel of 8 miRNAs and an algorithm was developed using the combined Discovery and Optimization Cohorts, with an AUC of > 0.90. When validated in the independent Validation Cohort, the panel demonstrated an AUC of > 0.85. Conclusions: We developed and validated a serum miRNA panel that is both sensitive (~70%) and specific (~90%) in detecting early-stage breast cancer.