Constructing metagenome-assembled genomes (MAGs) from complex metagenomic samples involves a series of bioinformatics operations, each requiring deep bioinformatics knowledge. Here, we present a protocol for constructing MAGs and conducting functional profiling to address biological questions. We describe steps for system configuration, data downloads, read processing, removal of human DNA contamination, metagenomic assembly, and statistical quality assessment of the final assembly. Additionally, we detail procedures for the construction and refinement of MAGs, as well as the functional profiling of MAGs.