Soil amino sugars have been widely used to evaluate the potential roles of microbes in mediating soil carbon (C) cycling and various pretreatment methods were used for its extraction. However, few studies assessed their potential influences on the soil amino sugar extraction. In this study, we investigated the effects of sample storage method and grinding on amino sugar extraction across different climatic zone and land uses. Results showed that the concentrations of soil amino sugars varied greatly among sample pretreatments and their impacts were highly dependent on climatic condition and land use. Specifically, higher concentrations of amino sugars were extracted from field-moist samples than dried samples in subtropical grassland, temperate forest and arable land with no significant differences among storage methods for the samples from subtropical forest, arable land, and temperate grassland. Moreover, grinding improved the extraction efficiency of amino sugars for the dried soils. Due to the reduced extraction concentration in dried soils, field-moist samples were recommended in priority. For the dried soils used for the long-term storage, grinding can be an option to improve the extraction efficiency. Such information will be valuable for reducing the uncertainty and improving the accuracy during the determination of soil amino sugars.