Objective To investigate the effect of folic acid and vitamin B12 on the efficacy of chemotherapy in patients with non-small cell lung cancer. Methods A total of 114 patients with non-small cell lung cancer who received chemotherapy in our hospital from May 2012 to February 2016 were selected, and divided into observation group (folic acid and vitamin B12+chemotherapy) and control group (conventional chemotherapy) according to the random table method. The clinical curative effect, ECOG score, high-sensitivity C-reactive protein (hs-CRP), carcinoembryonic antigen (CEA), CD4+/CD8+ and adverse reactions were compared between the two groups. Results In the 57 cases of the observation group, 6 cases had complete remission, 20 cases had partial remission, 25 cases were stable, and 6 cases progressed, the stability rate was 89.47%, the remission rate was 45.61%; in the control group, 3 cases completely relieved, 11 cases partially relieved, 23 cases were stable, and 20 cases had disease progression, the stability rate was 64.91% and the remission rate was 24.56%. The stability and remission rates of the two groups were statistically different between the two groups (P < 0.05). The ECOG scores of the two groups were statistically different (P < 0.05). There was no significant difference in hs-CRP, CEA or CD4+/CD8+ between the two groups before treatment (P > 0.05). After treatment, the levels of hs-CRP, CEA and CD4+/CD8+ in the two groups were significantly different (P < 0.05). The incidences of adverse reactions in the two groups were statistically different (P < 0.05). Conclusions Folic acid combined with vitamin B12 can effectively improve the effect of chemotherapy in patients with non-small cell lung cancer, improve the immune function and hematological parameters, and reduce the incidences of adverse reactions.