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Autoantibodies (AAbs) targeted tumor‐associated antigens (TAAs) have the potential for early detection of breast cancer. Here, 574 early‐stage breast cancer (ES‐BC) patients containing 4 subtypes (Luminal A, Luminal B, HER2+, TN), 126 benign breast disease (BBD) patients, and 199 normal healthy controls (NHC) were separated into three‐phases to discover, verify, and validate AAbs. In discovery phase using high‐throughput protein microarray, 37 AAbs with sensitivity of 31.25%‐86.25% and specificity over 73% in ES‐BC, and 40 AAbs with different positive rates between subtypes were identified as candidates. In verification phase, 18 AAbs were significantly increased compared with the Control (BBD and NHC) in focused array. Ten out of 18 AAbs exhibited a significant difference between subtypes ( P  < .05). In ELISA validation phase, 5 novel AAbs (anti‐KJ901215, ‐FAM49B, ‐HYI, ‐GARS, ‐CRLF3) exhibited significantly higher levels in ES‐BC compared with BBD/NHC ( P  < .05). The sensitivities of individual AAb and a 5‐AAbs panel were 20.41%‐28.57% and 38.78%, whereas the specificities were over 90% and 85.94%. Simultaneously, 4 AAbs except anti‐GARS differed significantly between TN and non‐TN subtype ( P  < .05). We constructed 3 random forest classifier models based on AAbs to discriminant ES‐BC from Control or BBD, and to discern TN subtype, which yielded an area under the curve of 0.870, 0.860, and 0.875, respectively. Biological interaction analysis revealed 4 TAAs, except for KJ901215, that were associated with well known proteins of BC. This study discovered and stepwise validated 5 novel AAbs with the potential to diagnose ES‐BC and discern TN subtype, indicating easy‐to‐detect and minimally invasive diagnostic value of serum AAbs ahead of biopsy for future application.

High‐throughput and multi‐phases identification of autoantibodies in diagnosing early‐stage breast cancer and subtypes