Objective To analyze the association between matrix metalloproteinase tissue inhibitor 3 (TIMP-3), DNA methyltransferase 1 (DNMT1) and the disease progression and prognosis of breast cancer patients. Methods A retrospective analysis was conducted on the clinical data of 102 breast cancer patients admitted to Xinyang Central Hospital from March 2022 to March 2024. The correlations between TIMP-3, DNMT1 and tumor markers in breast cancer patients were analyzed, as well as their predictive efficacy for adverse prognosis of breast cancer. Results The expression score of TIMP-3 in the late stage group was lower than that in the middle stage group, and the expression score of DNMT1 in the early stage group was higher than that in the middle stage group and the early stage group (P<0.05). The levels of carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA153), carbohydrate antigen 125 (CA125), carbohydrate antigen 19-9 (CA199) and tissue polypeptide antigen (TPA) in the late group were higher than those in the middle group and early group (P<0.05). TIMP-3 was negatively correlated with CEA, CA153, CA125, CA199 and TPA (P<0.05). DNMT1 was positively correlated with CEA, CA153, CA125, CA199 and TPA (P<0.05). The poor prognosis group had a significantly lower expression score of TIMP-3 and a significantly higher expression score of DNMT1 than the good prognosis group (P<0.05). The AUC value of TIMP-3 and DNMT1 combined detection in predicting poor prognosis of breast cancer was higher than that of TIMP-3 or DNMT1 alone. Conclusion The combined detection of TIMP-3 and DNMT1 can serve as an important auxiliary method for identifying patients with poor prognosis.