To investigate the regulatory effects of fertile topsoil layer construction (FLC) on rhizosphere microbial communities in saline cotton fields of Xinjiang, two cotton cultivars, Zhongmiansuo 1813 and Zhongmian 9001, were selected for field experiments. Using 16S rRNA and ITS amplicon sequencing, the effects of FLC and conventional fertilization treatment (CK) on cotton yield, rhizosphere soil properties, and microbial community composition were compared. The results showed that FLC significantly increased soil organic matter, alkali-hydrolyzable nitrogen, available phosphorus, and available potassium contents in the rhizosphere soil, resulting in seed cotton yield increases of 19.5% and 12.1% for Zhongmiansuo 1813 and Zhongmian 9001, respectively. FLC significantly altered rhizosphere microbial community structure, characterized by decreased bacterial α-diversity, whereas fungal Shannon diversity significantly increased under the FLC1813 treatment. Community composition analysis showed that FLC treatment promoted the enrichment of multiple dominant functional microbial taxa, with Bacillaceae and Salinimicrobium being significantly enriched in the FLC1813 treatment. Moreover, the two salt-tolerant cotton cultivars exhibited distinct rhizosphere microbial response patterns under the FLC system. The relative abundance of Salinimicrobium was higher and Fusarium abundance was lower in FLC1813, whereas Aspergillus and Fusarium tended to co-enrich in FLC9001. Integrated analyses based on LefSe (Linear discriminant analysis Effect Size), random forest modeling, and cross-kingdom co-occurrence networks identified Salinimicrobium as an important potential keystone taxon responding to FLC system. This genus acted not only as an important indicator taxon distinguishing different treatments but also as a key connector in the cross-domain co-occurrence network. The results indicate that FLC treatment can improve the rhizosphere soil environment in saline-alkali cotton fields and exhibit distinct microbial assembly patterns; different salt-tolerant cotton varieties exhibit distinct rhizosphere microbial response characteristics under the FLC system. This study provides a theoretical foundation for the precise regulation of the rhizosphere microecology in reclaimed saline-alkali farmland, the breeding of salt-tolerant cotton varieties, and the sustainable production of saline-alkali cotton fields.