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The study attempted to investigate the clinical value of serum cytokine levels in diagnosing colorectal cancer. Peripheral blood samples were obtained from 56 patients with colorectal cancer (colorectal cancer group) and 25 healthy subjects (control group) diagnosed in the Pathology Department at Shanxi Cancer Hospital between April 2020 and November 2020. The levels of 14 serum cytokines (interleukin (IL)-1β, L-17 A, IL-10, IL-4, IL-5, IL-6, IL-8, IL-22, IL-33, IL-18, IL-2RA, tumor necrosis factor (TNF)-α, TNF-β, and interferon (IFN)-γ) were analyzed using AimPlex flow cytometry high-throughput multi-factor detection technology. IL-2RA and IL-6 levels were significantly higher in the serum of patients with colorectal cancer than in the control group (P < 0.01), and the difference was of high statistical significance. IFN-γ, IL-8, and IL-5 levels were generally higher in the colorectal cancer group than in the control group (P < 0.05), and this difference exhibited statistical significance. The logistic regression model revealed that the colorectal cancer risk increased by 4.66 times for each increase in IL-6 (pg/mL) (P < 0.01), and by 3.01 times for each increase in IFN-γ (pg/mL) (P < 0.05). The Bayesian Kernel Machine Regression model indicated that the colorectal cancer risk increased by 0.95 times when all cytokines were at 75% compared to when at 50%. Furthermore, when the remaining cytokines were exposed at P50, the single factor exposure model showed that the levels of IL-6, IFN-γ, and TNF-β increased from P25 to P75. In contrast, the risk of colorectal cancer associated with these cytokines increased by 0.29 times, 0.248 times, and 0.4919 times. Meanwhile, a positive association was observed between the mixed effects of the 14 cytokines and the occurrence of colorectal cancer. This study's multi-method analysis demonstrated that colorectal cancer patients had significantly higher serum levels of IL-2RA and IL-6 (P < 0.01) and elevated IFN-γ, IL-8, and IL-5 levels (P < 0.05). Multivariate logistic regression showed that IL-6 (P < 0.01) and IFN-γ (P < 0.05) were strongly associated with disease risk (1-unit Ln increase raised the risk by 4.66- and 4.01-fold, respectively). RCS confirmed that IL-6 (with/without Ln transformation) and Ln-transformed IL-10 correlated positively with incidence (all P < 0.05). BKMR indicated the 14-cytokine mixed exposure at the 75th vs. the 50th percentile increased risk by 0.95. The mixed effect of the 14 cytokines detected using the mixed detection method positively correlated with the occurrence of colorectal cancer. These findings provide novel diagnostic insight for colorectal cancer.