In order to provide the scientific bases for the precise determination of soil microbial biomass (MBC), 9 chloroform fumigation time and 2 K₂SO₄ concentrations were performed to explore the optimal fumigation time and extracted concentration for alkaline soil MBC determination. The results showed that in alkaline soils, fumigation time is significantly correlated with soil organic matter (SOM) concentration. Higher SOM concentration needs longer fumigation time to obtain stable MBC content. Fumigation time for high SOM (≥60g/kg) is at least 24 hours, while that of middle and low SOM (≤30g/kg) is at least 18 hours. SOM concentration influences K₂SO₄ extraction efficiency, the high and middle SOM concentration soils need 0.5 mol/L K₂SO₄ solution to extract, 0.25 mol/L K₂SO₄ solution is enough to extract the MBC contents for low SOM soil. Thus, for a given soil with unknown SOM concentration, the recommended fumigation time and K₂SO₄ concentration is at least 24 h and 0.5 mol/L separately.