The mildew of stored grain will affect the safety of food，which can be monitored by the method of detecting the change of carbon dioxide (CO₂) concentration in grain bulk. As an indirect method, it is necessary to analyze comprehensively the related factors of grain bulk and the parameters of CO₂ sensor, which cansignificantly affect the detection value. The results showed that the mould count in the initial stage of the stored grain fungi growth was synchronized with the change of CO₂ concentration at different temperatures, and the correlation coefficient was greater than 0.99. CO₂ production decreased significantly at low temperature, the CO₂ produced at 30 ℃ was 4.7 times higher than that at 20 ℃ in the grain bulk of similar mould count. CO₂ gas diffusion was significantly affected by grain porosity and other factors. The porosity of rice was 30% higher than that of wheat, but the peak concentration of grain pile at the monitoring point 50 cm from the source of CO₂ was 50% higher than that of wheat pile, with 4 days earlier of the peak time. The monitoring point of 3 m below surface of grain bulk ccould reduce the effect of external gas exchange, and the monitoring effectiveness was better than that of the monitoring point with depth of 1 m when mildew position at 1 m or 2 m from grain bulk surface. The gas flow velocity and sampling quantity should be determined in the process of gas detection, and the detection value ccould be significantly reduced when the gas flow velocity exceeded the specified flow rate, and the gas sampling volume should be 1.5 times of the theoretical volume in the pipeline. Therefore, the carbon dioxide detection method by setting parameters reasonably could sensitively monitor the activity of mould in stored grain, and this was practically valuable for high efficiency in preventing and controlling the damage of mould.