Objective To investigate the changes in serum levels of thymidine kinase 1 (TK1), lemur tyrosine kinase 3 (LMTK3), and Carbohydrate antigen 199 (CA199) during adjuvant chemotherapy for colorectal cancer (CRC) and their value in evaluating the efficacy of chemotherapy. Methods Eighty CRC patients who underwent surgical treatment in Shangqiu Municipal Hospital from July 2021 to July 2023 were retrospectively selected for the study, and all of them received adjuvant chemotherapy 1 month after surgery, and according to the effect of chemotherapy after 3 cycles, the patients were classified into chemotherapy-effective group (33 cases) and chemotherapy-ineffective group (47 cases). The levels of serum TK1, LMTK3 and CA199 before chemotherapy, after one cycle of chemotherapy and after two cycles of chemotherapy were compared between the two groups. The correlation between serum indicators and chemotherapy effect was analyzed, and the evaluation value of combined detection of indicators for chemotherapy ineffectiveness in patients with CRC was analyzed. Results The levels of serum TK1, LMTK3, and CA199 in the chemotherapy-ineffective group were higher than those in the chemotherapy-effective group, and the levels of serum TK1, LMTK3, and CA199 gradually decreased with the progress of treatment, with statistically significant differences (P<0.05). After one cycle of chemotherapy and two cycles of chemotherapy, the levels of serum TK1, LMTK3, and CA199 were negatively correlated with the efficacy of chemotherapy, and the differences were statistically significant (P<0.05). After two cycles of chemotherapy, the area under the receiver operating characteristic (ROC) curve (AUC) of the combined prediction of serum TK1, LMTK3, and CA199 levels for predicting ineffective chemotherapy in CRC patients was higher than that of each individual indicator, and the difference was statistically significant (P<0.05). Conclusion The serum levels of TK1, LMTK3 and CA199 in patients with CRC have significant changes during the course of chemotherapy, which can provide an effective reference for clinical evaluation of chemotherapy effect.