Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. By using microcalorimetry, the thermokinetic method was developed to kinetic studies on Na+/K+-ATPase for the first time. Compared with other ones, the method provided accurate measurements of not only thermodynamic data but also the kinetic data. At 310.15 K and pH=7.4, the molar reaction enthalpy rHm was measured as (-40.408±1.9) kJ·mol-1. The Michaelis constant Km was determined to be (0.479±0.020)×10-3 mol·L-1 and consistent with literature figure which is about 0.5×10-3 mol·L-1. The maximum velocity Vmax obtained was (0.681±0.026) 靘ol Pi·min-1·mg protein-1. All of the data have good repeatability and self-consistency. The reliability of thermokinetic method was verified by the experimental results and further confirmed by colorimetric studies. Moreover, the effect of enzyme pre-dilution on its activities was also investigated. The use of microcalorimetry, the thermokinetic method was developed to kinetic studies on Na + / K + -ATPase for the first time. Compared with other ones, the method provided At 310.15 K and pH = 7.4, the molar reaction enthalpy rHm was measured as (-40.408 ± 1.9) kJ · mol -1. The Michaelis constant Km was determined to be ( 0.479 ± 0.020) × 10-3 mol·L-1 and consistent with the literature figure which is about 0.5 × 10-3 mol·L-1. The maximum velocity Vmax was was (0.681 ± 0.026) 靘 ol · min-1 · Mg protein-1. All of the data have good repeatability and self-consistency. The reliability of thermokinetic method was verified by the experimental results and further confirmed by colorimetric studies. Moreover, the effect of enzyme pre-dilution on its activities was also investigated.