Urease is a key enzyme affecting microbial metabolic activity, and its activity is affected by various pollutants,and pollutants have more complex effects on the activity of microbial urease. This study researched the effect of calcium and magnesium ions in landfill leachate on urease activity of anaerobic microorganism according to designing three kinds of calcium and magnesium ion mixtures with different concentration ratios by direct average ray method, investigating the concentration-effect curves of calcium and magnesium ions by BiPhasic and Logistic functions, analyzing the interaction relationship between calcium magnesium ions and microbial urease activity by equivalent line graphic method. The results indicated that with the increase of calcium concentration, the inhibitory effect of urease activity showed a trend of falling first and then rising, and the inhibitory effect of magnesium ions on urease activity increased with the increase of magnesium concentration. The inhibitory effect of calcium magnesium ions on urease activity was obviously time-dependent. The interaction of calcium magnesium ions mixed pollutants on urease activity was synergistic in the 1-4 reaction cycles, and the interaction was antagonistic in the 5 reaction cycles according to the equivalent line diagram. This paper provided support for further study on the action mechanism of pollutants on microbial urease by studying effect of calcium and magnesium ions in landfill leachate on urease .