Background: Plasma amyloid-beta (A beta) may facilitate identification of individuals with brain amyloidosis. Gut microbial dysbiosis in Alzheimer's disease (AD) is increasingly being recognized. However, knowledge about alterations of gut microbiota in preclinical AD, as well as whether the combination of plasma A beta and gut microbiota could identify preclinical AD, remains largely unknown. Methods: This study recruited 34 A beta-negative cognitively normal (CN-) participants, 32 A beta-positive cognitively normal (CN+) participants, and 22 patients with cognitive impairment (CI), including 11 patients with mild cognitive impairment (MCI) and 11 AD dementia patients. All participants underwent neuropsychological assessments and fecal microbiota analysis through 16S ribosomal RNA (rRNA) Illumina Miseq sequencing technique. Meso Scale Discovery (MSD) kits were used to quantify the plasma A beta(40), A beta(42), and A beta(42)/A beta(40) in CN- and CN+ participants. Using Spearman's correlation analysis, the associations of global standard uptake value rate (SUVR) with altered gut microbiota and plasma A beta markers were separately evaluated. Furthermore, the discriminative power of the combination of gut microbiota and plasma A beta markers for identifying CN+ individuals was investigated. Results: Compared with the CN- group, the CN+ group showed significantly reduced plasma A beta(42) (p = 0.011) and A beta(42)/A beta(40) (p = 0.003). The relative abundance of phylum Bacteroidetes was significantly enriched, whereas phylum Firmicutes and class Deltaproteobacteria were significantly decreased in CN+ individuals in comparison with that in CN- individuals. Particularly, the relative abundance of phylum Firmicutes and its corresponding SCFA-producing bacteria exhibited a progressive decline tendency from CN- to CN+ and CI. Besides, the global brain A beta burden was negatively associated with the plasma A beta(42)/A beta(40) (r = -0.298, p = 0.015), family Desulfovibrionaceae (r = -0.331, p = 0.007), genus Bilophila (r = -0.247, p = 0.046), and genus Faecalibacterium (r = -0.291, p = 0.018) for all CN participants. Finally, the combination of plasma A beta markers, altered gut microbiota, and cognitive performance reached a relatively good discriminative power in identifying individuals with CN+ from CN- (AUC = 0.869, 95% CI 0.782 similar to 0.955). Conclusions: This study provided the evidence that the gut microbial composition was altered in preclinical AD. The combination of plasma A beta and gut microbiota may serve as a non-invasive, cost-effective diagnostic tool for early AD screening. Targeting the gut microbiota may be a novel therapeutic strategy for AD.