A protocol to coextract the microbial metagenomic DNA and RNA from deep-sea sediment was developed for the microbiological study of environmental samples. The obtained pure metagenomic DNA with the size larger than 23 kb and stable RNA could be used directly for PCR and reverse transcription-PCR (RT-PCR) respectively. The direct lysis including the treatments of SDS, proteinase and lysozyme was applied to acquiring the metagenomic DNA and RNA furthest. Prior to the lysis treatment, the glass bead and denaturing solution were added to enhance the lysis efficiency and keep the integrity of RNA respectively. Denaturing gradient gel electrophoresis (DGGE) was applied in accessing the microbial 16S rRNA diversity by PCR and RT-PCR amplification from a single extraction. The pattern obtained by this analysis revealed some differences between them, indicating the efficiency of the protocol in extracting the metagenomic DNA and total RNA from deep-sea sediment.