Updated project metadata. We developed a free-flow isoelectric focusing (FFIEF) based metaproteomics workflow to reduce the host interferences and enrich the low-abundant bacteria for better interpretation of salivary microbiome. We firstly tested the pretreatment module that could significantly reduce the host interferences by differential centrifugation and filtration. Then the FFIEF module was applied to separate the microbes and enrich the low-abundant bacteria, which showed a significant improvement in the identification efficiency of microbiome sample. Applying our method to lung cancer, we successfully identified Fusobacterium, Neisseriaceae, Actinomycetaceae, Burkholderiales, and Spirochaetia were associated with lung cancer as previous sequencing studies did, along with other 16 significantly different species. The dysregulated functions in lung cancer microbiome were also explained in details. Our workflow provides improved efficiency in identification and characterization of the salivary microbiome with great reproducibility. The ability of enriching low-abundant bacteria and functions enables in-deep analysis of previously underestimated information.