This study introduces a novel mathematical and bioinformatics framework for constructing ecological association networks, named molecular ecological networks (MENs), using Random Matrix Theory (RMT)-based methods. The approach is designed to automatically define network structures and robustly handle noise, making it suitable for high-throughput metagenomics data. The authors applied this method to analyze microbial communities subjected to long-term experimental warming based on 16S rRNA gene pyrosequencing data. The constructed MENs exhibited scale-free, small-world, and modular topological features, consistent with previous studies on molecular ecological networks. Eigengene analysis revealed that eigengenes represented module profiles well. Environmental traits such as temperature and soil pH were found to be important in determining network interactions. To facilitate the application of this method, the authors developed an open-access Molecular Ecological Network Analysis Pipeline (MENAP). The RMT-based molecular ecological network analysis provides powerful tools for elucidating network interactions in microbial communities and their responses to environmental changes, which are crucial for microbial ecology and environmental microbiology research.This study introduces a novel mathematical and bioinformatics framework for constructing ecological association networks, named molecular ecological networks (MENs), using Random Matrix Theory (RMT)-based methods. The approach is designed to automatically define network structures and robustly handle noise, making it suitable for high-throughput metagenomics data. The authors applied this method to analyze microbial communities subjected to long-term experimental warming based on 16S rRNA gene pyrosequencing data. The constructed MENs exhibited scale-free, small-world, and modular topological features, consistent with previous studies on molecular ecological networks. Eigengene analysis revealed that eigengenes represented module profiles well. Environmental traits such as temperature and soil pH were found to be important in determining network interactions. To facilitate the application of this method, the authors developed an open-access Molecular Ecological Network Analysis Pipeline (MENAP). The RMT-based molecular ecological network analysis provides powerful tools for elucidating network interactions in microbial communities and their responses to environmental changes, which are crucial for microbial ecology and environmental microbiology research.