Abstract: Unlike the traditional enrichment, separation and purification technology to obtain some purely cultured pollutant-degrading functional microorganisms, stable isotope technology (SIP-DNA) can more easily identify functional genes. In this study, the core technology of density gradient centrifugation in SIP-DNA was used to obtain the conditions and related parameters of the technology. The buoyancy density and DNA concentration results show that this method can effectively isolate isotope-labeled and non-isotopic-labeled DNA and lay the foundation for subsequent high-throughput sequencing of SIP-DNA. It also provides technical means for centrifuge managers and experimenters to use the method correctly.