The testing efficiency of ELISA analyzer is seriously affected the timeconsuming incubation and the accuracy will be reduced by the differences in the degree of reagent reaction due to temperature instability. Aiming at these problems, a method of segmentation incubation based on the improved digital PID algorithm was proposed. Firstly, the temperature characteristics of reagent and incubation system were analyzed and the parameters in preheating and constant temperature stage were determined. Secondly, different algorithms were applied in the different sections of incubation process according to the parameters and control indicators. And then, the experiment system was set based on ELISA analyzer framework to verify the time of temperature rising and stability of reagent. The experiment results indicate that the reagent is heated up from 21 ℃ to 37.5 ℃ in 239±5 seconds, meanwhile, the temperature deviation of isothermal segment is less than ±0.2 ℃. The method proposed in this paper achieves fast and stable temperature control and ensures enzyme reaction fast and stable.