Objective:To investigate the levels of serum high mobility group box (HMGB) 2 and HMGB3 and clinical significance in patients with non-small cell lung cancer (NSCLC) .Methods:A total of 137 NSCLC patients admitted to the First Affiliated Hospital of Xi'an Medical University from January 2020 to January 2022 were selected as the NSCLC group, and another 90 cases who underwent healthy medical checkups during the same period were selected as the healthy group. Serum HMGB2 and HMGB3 levels were compared between the two groups. The relationship between serum HMGB2 and HMGB3 levels and the clinical and pathological characteristics of NSCLC patients was analyzed. NSCLC patients were divided into a good prognosis group ( n=86) and a poor prognosis group ( n=51) according to the prognosis, and the clinical data of the two groups were compared. Multivariate logistic regression was used to analyze the influencing factors of the prognosis of NSCLC patients. Receiver operator characteristic (ROC) curve was used to analyze the predictive value of serum HMGB2 and HMGB3 on the prognosis of NSCLC patients. Results:Serum HMGB2 [ (6.35±1.66) ng/ml vs. (2.58±0.76) ng/ml, t=20.19, P<0.001] and HMGB3 [ (2.48±0.56) ng/ml vs. (1.09±0.13) ng/ml, t=23.13, P<0.001] levels in NSCLC group were higher than those in healthy group. Serum HMGB2 and HMGB3 levels of NSCLC patients with a history of smoking ( t=2.80, P=0.006; t=5.04, P<0.001), lymph node metastasis ( t=3.53, P=0.001; t=4.02, P<0.001), and TNM stage Ⅲ-Ⅳ ( t=2.58, P=0.011; t=3.82, P<0.001) were significantly higher than those of patients with no history of smoking, no lymph node metastasis, and TNM stage Ⅰ-Ⅱ. The serum levels of HMGB2 [ (7.80±1.83) ng/ml vs. (5.49±1.56) ng/ml, t=7.85, P<0.001] and HMGB3 [ (2.91±0.78) ng/ml vs. (2.23±0.43) ng/ml, t=6.58, P<0.001) ] in the poor prognosis group were higher than those in the good prognosis group, and the proportion of patients with lymph node metastasis ( χ2=4.81, P=0.028), history of smoking ( χ2=11.67, P=0.001), and TNM stage Ⅲ-Ⅳ ( χ2=6.18, P=0.013) was significantly higher than that in the good prognosis group. Multivariate logistic regression analysis showed that lymph node metastasis ( OR=1.96, 95% CI: 1.14-3.36, P=0.015), smoking history ( OR=2.02, 95% CI: 1.33-3.06, P=0.001), TNM stage ( OR=2.28, 95% CI: 1.35-3.86, P=0.002), HMGB2 ( OR=2.01, 95% CI: 1.40-2.91, P<0.001), and HMGB3 ( OR=1.99, 95% CI: 1.25-3.15, P=0.003) levels were independent influencing factors of prognosis of NSCLC patients. ROC curve analysis showed that the area under the curve (AUC) of serum HMGB2 and HMGB3 alone and in combination to predict the prognosis of NSCLC patients were 0.833, 0.862 and 0.922, respectively, and the AUC predicted by the combination was significantly higher than that predicted by serum HMGB2 ( Z=2.44, P=0.015) and HMGB3 ( Z=2.54, P=0.011) alone. Conclusion:Serum HMGB2 and HMGB3 levels are up-regulated in NSCLC patients and are closely associated with poor prognosis, and the combined detection of the two has certain predictive efficacy for prognosis of NSCLC patients.