<p>Accurate estrus detection is critical for optimizing reproductive efficiency in beef cattle. This study characterized blood metabolites and cervical microbiota associated with estrus in Simmental cattle using an integrated multi-omics approach. Twelve multiparous cows were assigned to estrus (<i>n</i> = 6) or nonestrus (<i>n</i> = 6) groups based on behavioral, hormonal, and ultrasonographic criteria. Serum samples were analyzed via ELISA and UPLC-MS/MS, while cervical microbiota were profiled using 16S rRNA and ITS sequencing. Serum biochemical analysis revealed elevated levels of total protein, albumin, glucose, and insulin-like growth factor-1 during estrus (<i>p</i> &lt; 0.01). Untargeted metabolomics identified 258 differentially abundant metabolites enriched in amino acid and lipid metabolism pathways. Cervical microbiota analysis showed that <i>Firmicutes</i> and <i>Bacteroidetes</i> were the dominant bacterial phyla, with <i>Bacteroidetes</i> increasing by 13.36% during estrus. Fungal <i>Ascomycota</i> dominated the estrus group (78.38% vs. 67.20%), accompanied by reduced alpha diversity (<i>p</i> &lt; 0.05). LEfSe analysis identified 22 bacterial and 16 fungal biomarkers associated with estrus. Correlation analysis revealed that E2 was positively correlated with <i>Bacteroidota</i> (<i>r</i> = 0.604) and fungal genera <i>Bacteroides</i> (<i>r</i> = 0.703), while progesterone showed negative correlations. This study reveals a complex microbiota-metabolite-host network specific to estrus in Simmental cattle, providing potential biomarkers for estrus detection.</p> Graphical Abstract <p></p>