Background Hemophagocytic lymphohistiocytosis (HLH) is a category of immunological illnesses that cause out-of-control T cells and macrophages to release life-threatening cytokines. The HLH-2004 diagnostic criteria are the gold standard for HLH diagnosis, but there is a need to investigate the usefulness of various cytokines for HLH diagnosis. Methods Patients admitted to Beijing Friendship Hospital of Capital Medical University from January 2016 to December 2020 were included in this retrospective study, with 166 patients with confirmed HLH and 142 febrile patients requiring differential diagnosis completing the sum. Multiplex cytokine assays using multifactor liquid phase microarray technology-based multifactor liquid phase microarray technology were used to detect 33 cytokines. Twenty-eight cytokines detected using the Luminex analytical platform technology were ultimately included in the analysis. Results Interleukin-1 receptor antagonist (IL-1 RA), IL-18, interferon-gamma (IFN-gamma), and interferon-induced protein 10 (IP-10) regulated upon activation normal T cell expressed and secreted (RANTES), eotaxin, growth-related oncogene alpha (GRO-alpha), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) were higher in the HLH group than in the non-HLH group, and the differences were statistically significant. Among them, the area under the curve (AUC) for IL-18 for HLH diagnosis was reported for the first time as 82.69%, with a sensitivity of 76.32% and a specificity of 79.61%; the AUC of IL-1 RA was 72.34%, with a sensitivity of 62.71% and a specificity of 75.97%; and the AUC of IP-10 was 71.73%, with a sensitivity of 60.14% and a specificity of 75.15%. Moreover, the AUC of the combined diagnostic tests for IL-1 RA, IL-18, IFN-gamma, IP-10, and RANTES was 99.6%, with a sensitivity of 95.8% and a specificity of 98.6%. Conclusion Our study concluded that multiple cytokines are valid biological markers for the diagnosis of HLH. The findings of this study remain to be validated in an external dataset.