Kawasaki disease (KD) is the most common cause of acquired heart disease in young children and has been suggested to be an immune-mediated condition, which was supported by our previous findings that FCGR2A, BLK, and CD40 are KD susceptibility genes. However, diagnosing KD can be difficult especially at early stage of the disease because of its varied clinical manifestations and lack of specific laboratory tests. The aim of the present study was to identify biomarkers that may aid in the early diagnosis of KD. We conducted a three-phase study: discovery, replication, and blinded validation. The 214 children with fever and clinical features suggestive of KD were recruited in Taiwan from separate geographic medical centers. By using an unbiased, large-scale, quantitative protein array, we globally analyzed the profiles of cytokines, chemokines, and cell adhesion molecules in their plasma samples. During the discovery phase [n (KD) = 37, n (control) = 20], the expression of one cell adhesion molecules, two chemokines, and three cytokines (Th-17-related), (named gene-1~6) were upregulated during the acute phase in KD patients compared to that in the controls. Receiver-operating characteristic analysis of the combined discovery and replication data [n (KD) = 77, n (control) = 77] showed that the level of gene-3 had high area under the curve values (AUC, 0.94). Using the optimal cut-off value to predict KD, the sensitivity and specificity of the gene-3 was found to be 90% and 87%, respectively. Blinded validation study also confirmed the excellent discrimination of gene-3 in identifying KD cases from non-KD cases among subjects who were highly suspected KD cases, including incomplete KD or febrile cases with scarlet fever, even in the very early stage (<4 days).With intravenous immunoglobulin treatment, levels of gene-3 returned to normal. Furthermore, we also found the downstream receptor of gene-3 was activated in the T cells of acute KD patients. Taken together, the newly identified biomarker, gene-3, may be useful in diagnosing KD and monitoring patients’ treatment responses. The selective increase of gene-3 in KD also reveals that a novel unique signal pathway involved in KD, which may provide a critical clue for further investigating the pathogenesis of KD.