The present study aimed to assess the kinetics of cytokine release and compare the accuracy of serum biomarkers for the diagnosis of macrophage activation syndrome (MAS) associated with Kawasaki disease (KD). Serum neopterin, interleukin (IL)-18, IL-6 and soluble tumour necrosis factor receptor type I (sTNFR-I) and sTNFR-II levels were determined using enzyme-linked immunosorbent assay in 78 patients with KD, including five with MAS. Results were compared to the clinical features of MAS. Serum neopterin, IL-18, sTNFR-II levels and sTNFR-II/I ratio were significantly elevated in KD patients with MAS compared to those in the acute phase. Receiver operating characteristic curve analysis revealed areas under the curve and cutoff values of neopterin, IL-18, sTNFR-II levels and sTNFR-II/I ratio were 0.9750/30.0 nmol/L, 0.9813/1165 ng/mL, 0.9969/16,600 pg/mL and 0.9875/4.475, respectively. Serum sTNFR-II levels correlated positively with disease activity. These findings indicate that overproduction of interferon (IFN)–γ and TNF-α reflected by increased serum levels of neopterin and sTNFR-II are closely associated with the pathogenesis of MAS associated with KD. Serum sTNFR-II levels might be a useful marker to diagnose the transition to MAS.