High-throughput sequencing at cellular level is a powerful tool for identifying active microbes in complex soil microbiome. Soil microcosms were constructed with three treatments including CK (control), moisture soil (60% soil water holding capacity) and flooded soil (100% soil water holding capacity) in a typical paddy soil in northeast China. The effects of soil water on active microbes were investigated based on soil microbial cell extraction technology and high-throughput sequencing. The findings of this study suggested that the increase in soil moisture content had a significant suppressive effect on soil microbial respiration activity. The cumulative CO₂ emissions in flooded soil accounted for 87.1% of that in moisture soil over a 48-h incubation period. The analysis of high-throughput sequencing further indicated that the predominant active microbiome was accurately identified at both the DNA and cellular DNA levels, which included the key phyla of Proteobacteria and Firmicutes, and key genus of Pseudomonas in CK, moisture and flooded paddy soil. However, the real-time qPCR and high-throughput sequencing consistently suggested that significant differences in microbial abundance, diversity and community structure were observed under different soil water conditions in paddy soil over a 48-h incubation period. The increase in soil moisture content had a significant stimulative effect on soil total microbial abundance, but a suppressive effect on soil active microbial diversity in the incubated paddy soil. The active dominant genus of Pseudomonas in moisture soil condition had a lower relative abundance (6.7%) than that in flooded soil (11.6%). The analysis of the microbial co-occurrence network further revealed that an increase in soil moisture content significantly increased the negative correlations between active phylotypes, thereby intensifying their competitive interactions in the paddy soil. The proportion of positive network correlations in moisture soil (86.2%) was higher than that in flooded soil (61.4%) after the incubation. Functional prediction based on 16S rRNA gene sequencing indicated that soil water had significant effects on the processes of biological nitrogen fixation and sulfur-related microbial metabolisms in the tested paddy soil. The trends observed for both the total and active microbiome at the DNA and cellular DNA levels were found to be similar over a 48-h incubation period. In total, these results indicated that the increase in soil water content significantly affected soil microflora and the function by strengthening the competitions between microbial communities in paddy soil. Therefore, appropriate reduction of soil water content in paddy fields is of great significance for the stability of microbial functions in northeastern paddy soils.