Background. M2 type Pyruvate kinase (PKM2) is the key rate-limiting enzyme of glycolysis, and it mainly exists as a dimer in tumor cells. This study aims to establish an enzyme kinetic assay for serum tumor M2 pyruvate kinase (TuM2-PK), and to evaluate its diagnostic value in breast cancer. Methods. The catalytic kinetics of Pyruvate Kinase (PK) was examined. Its allosteric regulation property and Michaelis constant were then measured. Next, the levels of TuM2-PK in serum were detected and compared with results from an enzyme-linked immunosorbent assay (ELISA). Finally, the levels of TuM2-PK among breast cancer patients, post-mastectomy patients, patients with benign breast diseases, and healthy controls were compared. Results. A PK kinetic assay was established in this study. The assay reaction time is 108 seconds, and the optimum pH level is 8.0. In the presence of the allosteric activator fructose 1, 6-bisphosphate (FBP), the Km of PK for phosphoenolpyruvate (PEP) is 0.15 mmol/L and the Vmax is 330 μmol/min. The levels of TuM2-PK in serum obtained by enzyme kinetics are comparable to the ELISA results. Both assays showed that TuM2-PK in breast cancer patients was increased from stage I to IV. Importantly, TuM2-PK levels were significantly different between early and late-stage breast cancer patients (stage I and stage II vs. stage III and IV), as well as between late-stage and non-malignant patients (p<0.05). No statistical difference was found between benign breast disease patients and healthy controls.Conclusions. An enzyme kinetic assay of serum TuM2-PK was successfully established and may be useful for breast cancer diagnosis.