Chikungunya (CHIKV) and Dengue (DENV) viruses cause an acute febrile illness which is hard to clinically differentiate and treat since both exhibit similar symptoms. Hence, this study was aimed at identifying the expression profiles of cytokines on co-infected samples and compare with CHIKV and DENV mono-infected samples. Serum samples of 292 suspected patients during 2009-2011 were analyzed. The presence of primary (IgM)/secondary (IgG) antibodies and early NS1 Dengue antigens were confirmed by capture ELISA. Molecular diagnosis and serotypes were discriminated by RT-PCR, confirmed by sequencing. All the plasma samples were assayed for cytokine expression by BDTM cytometry bead array (CBA) and compared with independent mono-infection viral load. Among the tested samples, 82 were confirmed as Dengue positive; 52 through IgM (17.8%), and 30 through IgG (10.2%). Additionally, 186 samples were confirmed as Chikungunya, 96 through IgM (32.6%) and 92 through IgG (31.5%) ELISA, respectively. Interestingly, 19 patients were co-infection positive in which, only 6 were confirmed for CHIKV and 7 for DENV by RT-PCR. Among 8 cytokines, IL-2, IL-8, IFNα, IFN γ, and IL-12 were found to be significantly different between co-infected and CHIKV mono-infected patients and correlated with viral load. DENV viral load was correlated with cytokine expression and a significant difference in IL-2 and IL-12 was observed between DENV mono-infected and DENV and CHIKV co-infected patients. Results indicated that apart from serological and molecular confirmation, cytokines could be used as a specific biomarker for the diagnosis of DENV and CHIKV. In the future, the role of independent cytokines can be determined to understand the pathogenesis and etiology of these dreadful diseases.