Objective To investigate the correlation between the levels of serum tripartite domain protein 27 (TRIM27), tumor necrosis factor-like weak apoptosis-inducing factor (TWEAK), and Lin-28 homolog B (Lin28B) and the pathological characteristics and clinical prognosis of endometrial cancer (EC) patients. Methods A total of 115 EC patients admitted to the Second People's Hospital of Jiaozuo from June 2021 to June 2024 were selected as the study group, and 115 healthy physical examination subjects in the same period as the control group. The levels of serum TRIM27, TWEAK and Lin28B mRNA were compared between the two groups. The levels of TRIM27, TWEAK and Lin28B mRNA in patients with different pathological characteristics and prognosis in the study group were compared. The correlation between serum TRIM27, TWEAK and Lin28B mRNA levels and pathological characteristics and prognosis was analyzed. The receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to analyze the diagnostic value of serum TRIM27, TWEAK and Lin28B mRNA for EC. Results The levels of serum TRIM27, TWEAK and Lin28B mRNA in the study group were higher than those in the control group (P<0.05). There were statistically significant differences in serum TRIM27, TWEAK and Lin28B mRNA levels in patients with different FIGO stages, tumor diameters and lymph node metastasis in the study group (P<0.05). The levels of serum TRIM27, TWEAK and Lin28B mRNA were positively correlated with FIGO stage, tumor diameter and lymph node metastasis (P<0.05). The AUC of serum TRIM27, TWEAK and Lin28B mRNA alone and combined diagnosis of EC was 0.712, 0.735, 0.685 and 0.857, respectively, and the combined AUC was the highest. The levels of serum TRIM27, TWEAK and Lin28B mRNA in patients with good prognosis were lower than those in the control group (P<0.05). Serum TRIM27, TWEAK and Lin28B mRNA levels were positively correlated with prognosis (P<0.05). Conclusion The high expression of TRIM27, TWEAK, and Lin28B mRNA in the serum of EC patients is closely related to their pathological characteristics and prognosis. TRIM27, TWEAK, and Lin28B can serve as auxiliary markers for diagnosing EC.