Objective To analyze the expression and clinical diagnostic value of serum heat shock protein 90α (Hsp90α), eosinophil chemotactic protein 2 (eotaxin-2), and tumor necrosis factor receptor associated factor 6 (TRAF6) in patients with early colorectal cancer. Methods From May 2019 to December 2020, 119 patients with stage Ⅰ and stage Ⅱ colorectal cancer diagnosed by postoperative pathology admitted to our hospital were consecutively included in the colorectal cancer group. After a 3-year follow-up, surviving patients were divided into a recurrence and metastasis group and a non-recurrence and metastasis group.Additionally, 121 patients with colorectal adenoma were collected as the benign lesion group. Enzyme-linked immunosorbent assay (ELISA) method was used to detect serum levels of Hsp90α, eotaxin-2, and TRAF6. If the level was higher than the median expression level among all patients, it was defined as high expression; otherwise, it was defined as low expression. Receiver operator characteristic (ROC) curve was used to analyze the diagnostic value of serum Hsp90α, eotaxin-2, and TRAF6 in distinguishing early colorectal cancer from benign lesions and in predic-ting recurrence and metastasis in colorectal cancer patients.Thearea under the curve (AUC) was comparedusing Z-test.Kaplan-Meier method was used to analyze the relationship between these indicators and 3-year survival rate of colorectal cancer patients. Results The serum levels of Hsp90α, eotaxin-2, and TRAF6 in the colorectal cancer group were higher than those in the benign lesion group (P<0.05). In colorectal cancer patients with a tumor diameter of ≥ 5 cm and TNM stage Ⅱ, the serum levels of Hsp90α, eotaxin-2, and TRAF6 were higher than in those with a tumor diameter of<5 cm and TNM stage Ⅰ (P<0.05). The AUC for differentiating early colorectal cancer from benign lesions using serum Hsp90α, eotaxin-2, and TRAF6 alone and in combination were 0.783 (95%CI: 0.723-0.843), 0.795 (95%CI: 0.739-0.851), 0.753 (95%CI: 0.690-0.816), and 0.906 (95%CI: 0.867-0.945), respectively. The combined AUC was higher than the individual AUC of each indicator (Z=3.411, 3.151, 4.054, P<0.05), with sensitivity values of 52.99%, 85.73%, 73.12%, and 86.68%, and specificity of 75.98%, 59.52%, 71.12%, and 82.62%, respectively.The diagnostic thresholdsfor Hsp90α, eotaxin-2, and TRAF6 were 178.27 ng/mL, 198.77 pg/mL, and 3.49 μg/mL, respectively. A total of 119 colorectal cancer patients were followed up for 3 years, with 92 surviving (including 14 survivors withrecurrence and metastasis) and 27 dying due to recurrence and metastasis. The 3-year survival rate of patients with high serum expression of Hsp90α, eotaxin-2, and TRAF6 were 62.07%, 61.67% and 65.57%, which were lower than that of patients with low expression, which were 91.80%, 93.22% and 89.66% (P<0.05). The serum levels of Hsp90α, eotaxin-2, and TRAF6 in colorectal cancer patients with recurrence and metastasis were higher than those in the non-recurrence and metastasis group (P<0.05). The AUC for predicting colorectal cancer recurrence and metastasis using serum Hsp90 α, eotaxin-2, and TRAF6 alone and in combination were 0.836 (95%CI: 0.760-0.911), 0.701 (95%CI: 0.597-0.804), 0.821 (95%CI: 0.741-0.900), and 0.968 (95%CI: 0.934-0.998), respectively. The combined AUC was higher than the individual AUC of each indicator alone (Z=3.073, 4.770, 3.351, P<0.05). Conclusion The serum levels of Hsp90α, eotaxin-2, and TRAF6 are higher in patients with early colorectal cancer, and they are associated with the patient’s prognosis. The combination of these three is expected to serve as clinical auxiliary diagnostic and prognostic markers.