Cellulasel plays an very important role in the ecosystem and the nutrient cycling. It can decomposite cellulose to small molecules of sugar and provide energy for biological activities. How to measure cellulase activity quickly and accurately is of great significance for the diagnosis and restoration for soil ecosystem. Different nitrogen treatments and cultivate would influenced enzyme activity. This study use automatic TECAN Infinite 200 Multi-Mode Microplate Reader (made in Germany) bombined with 96 microplates and fluorescence (4-Methylumbelliferyl β-D-cellobiopyranoside (Cel)) detection method to detect the activity of cellulasel under different tartary buckwheat (DiQing-low nitrogen toleration, HeiFeng 1-low nitrogen intoleration). During the process, the experiment use less substrate and put the control and sample together in the same plat,then reduced the culture space and operation time. The results showed that except the normal nitrogen treatment under the maturation stage, cellulasel was higher in DiQing than in HeiFeng 1 in the stage of seeding and flowering. In addition, the activity of cellulase in root trace of Diqing was higher than that of Heifeng 39.50% under low nitrogen treatment at seedling stage. Cellulasel had a significant difference among nitrogen treatments in the flowering and maturation stage, much higher activity in normal treatment when compared with the others under DiQing, but Heifeng only showed the same trend in flowering stage. The enzymatic activity of sterilizing treatment was significantly lower than that of normal nitrogen treatment of 54.29%, which indicated that some of cellulase in root trace of Diqing Tartary buckwheat originated from the contribution of microorganism, and the activity of microorganism was higher when the nutrient was sufficient. The analysis of cellulase activity by fluorescence spectrum showed that the resistant variety Diqing Tartary buckwheat could resist the stress of external environment by increasing the activity of soil enzyme in root trace. While in the poor nutrient soil in Loess Plateau, maybe selecting barren-tolerant varieties would reduce costs and increase the benefits for farmers. Compared with traditional method, fluorescence microplate fluorescence microplate method is quick, accurate and economical. It is also suitable for mass sample determination, which can provide timely data support for foreign precision agriculture production and soil testing formula fertilization in the further.