Objective To investigate the relationship between the expression of serum microRNA-326(miR-326)and microRNA-623(miR-623)in non-small cell lung cancer(NSCLC)patients and their clinical pathological characteristics and prognosis.Methods A total of 114 NSCLC patients diagnosed in our hospital from March 2019 to June 2020 were collected as study subjects as case group,123 healthy individuals who underwent physical examination were as the control group.According to the 3-year prognosis,patients were separated into a survival group of 71 cases and a death group of 43 cases.Patient related clinical data were collected,real-time fluorescence quantitative PCR method was applied to detect the expression levels of miR-326 and miR-623 in various serum samples;Kaplan-Meier method was applied to analyze the relationship between the expression levels of serum miR-326 and miR-623 in NSCLC patients and their 3-year prognosis;Cox proportional risk regression model was applied to analyze the influencing factors of 3-year prognosis in NSCLC patients.Results The expression levels of serum miR-326 in the case group and control group were 0.64±0.15 and 1.02±0.23,respectively,and the expression levels of miR-623 were 0.56±0.10 and 0.98±0.15,respectively.The difference between the two groups was statistically significant(P＜0.05).The proportions of patients with low expression of miR-326 and miR-623 in low differentiation,TNM stage Ⅲ+Ⅳ,and lymph node metastasis were higher than those in high differentiation,TNM stage Ⅰ and Ⅱ,and no lymph node metastasis(P＜0.05).The 3-year survival rates of patients with low expression of miR-326(20/55,36.36％)and miR-623(27/61,44.26％)in the serum of NSCLC patients were lower than those of patients with high expression of miR-326(51/59,86.44％)and miR-623(44/53,83.02％)(Log Rank x2=32.060,22.812,P＜0.05).Serum miR-326[(0.55±0.09)vs.(0.69±0.11)]and miR-623 levels[(0.48±0.08)vs.(0.61±0.10)]of patients in the death group were significantly lower than those in the survival group(P＜0.05).The area under the curve(AUC)for poor prognosis of serum miR-326 and miR-623 alone and in combination in patients diagnosed with NSCLC were 0.828(95％CI:0.754 to 0.901),0.763(95％CI:0.671 to 0.855),and 0.903(95％CI:0.849 to 0.958),respectively.The proportions of patients with TNM stage Ⅲ+Ⅳ,lymph node metastasis,low expression of miR-326 and low expression of miR-623in the death group were higher than those in the survival group(P＜0.05).MiR-326 and miR-623 were protective factors affecting 3-year mortality in NSCLC patients,while TNM staging and lymph node metastasis were independent risk factors affecting 3-year mortality in NSCLC patients(P＜0.05).Conclusion The low expression of miR-326 and miR-623 may be involved in the occurrence and development of lung cancer,which is closely related to the clinical pathological characteristics and poor prognosis of patients.