Abstract: Objective To investigate the relationship between expression of serum thymidine kinase 1 （TK1） and the efficacy of pemetrexed regimen in advanced non-small cell lung cancer （NSCLC）. Methods A total of 74 patients with advanced NSCLC diagnosed pathologically from January 2013 to January 2016 were enrolled in this study. All patients received pemetrexed monotherapy or platinum combination therapy. Peripheral blood samples from NSCLC patients were collected and serum levels of TK1 were detected by immunoblot enhanced chemiluminescence. The relationship between serum TK1 levels and clinical pathological parameters （sex， age， tumor size， differentiation， clinical stage， smoking history and lymph node metastasis） was analyzed. Meanwhile， peripheral blood samples from 88 healthy subjects were selected as controls. The RECIST1.1 version was used to evaluate the short-term effects of chemotherapy and the prognosis was followed up. The relationship between serum TK1 levels and prognosis was evaluated. Results TK1 level was （3.677±0.172） pmol／L （median： 3.665 pmol／L； four quantile： 2.618-4.423 pmol／L） in the NSCLC group， and （1.510± 0.072） pmol／L （median： 1.530 pmol／L； four quantile： 0.950-2.010 pmol／L）. The TK1 level was higher in NSCLC group than in control group and there was statistical significant differences （P＜0.05）. TK1 levels were not related to sex， age， smoking history and lymph node metastasis of NSCLC， but were related to tumor size， differentiation type and TNM stage. The serum level of TK1 was （4.362±0.210） pmol／L in tumor over 3 cm in size， higher than （2.914±0.217） pmol／L in tumor less than 3 cm in size （P＜0.05）. The serum TK1 level of the patients with low differentiation was （4.865±0.196） pmol／L， higher than （2.867±0.171） pmol／L of the high and middle differentiation （P＜0.05）. The serum TK1 level of TNM stage Ⅳ was （4.550±0.187） pmol／L， higher than （3.012±0.218） pmol／L of stage Ⅲ （P＜0.05）. The TK1 level of patients with chemotherapy sensitivity was （1.991±0.199） pmol／L （median： 2.360 pmol／L； four quantile： 1.360-2.560 pmol／L）， and of patients with chemotherapy resistance was （4.259±0.158） pmol／L （median： 4.250 pmol／L； four quantile： 3.570-4.870 pmol／L）. The sensitive TK1 level was higher in resistance group than in sensitive group （P＜0.05）. The progression-free survival of high-level TK1 （＞3.665 pmol／L） was 6.6 months， shorter than 8.5 months of low level of TK1 （P＜0.05）. Conclusion There was high expression of TK1 in serum of NSCLC patients， and tumor size， differentiation types and clinical stages were associated with higher TK1. The total effective rate is lower and shorter PFS in patients with higher level of TK1， suggesting a role in NSCLC development.