The invention discloses a method for simultaneously measuring the ammonia oxidation rate and the nitrite oxidation rate based on a mathematical model. Without adding any inhibitor, the water sample orsediment sample was cultured in situ without light, and a culture system was established. The concentration of DIN(NH4+, NO2-and NO3-) in water of the culture system is measured continuously, and a time sequence of DIN concentration change is obtained. A Boltzmann model is used to fit the DIN time series of water body, and the fitting mathematical equation is obtained. On this basis, the fittingmathematical equation is first-order differentiated by MATLAB, and the instantaneous velocity trend diagram can be obtained by the differential equation. At the same time, the average reaction rate iscalculated based on the model. This method overcomes the disturbance of inhibitor dependence, obtains both the instantaneous reaction rate and the average reaction rate of Va and Vn in one system, presents the kinetic process of microbial nitrification in the environment accurately, and provides a convenient and accurate method for the research in this field.