Recently, researches of molecular biology for the identification of root-knot nematode (RKN) species have been reported in plant quarantine. In this study, applicable and reproducible method to extract high quality genomic DNA from single nematode (Meloidogyne spp.) was developed. Also, the modified method was verified by DNA manipulation techniques such as PCR amplification and cloning. Single juvenile was floated in a drop of water and digested with proteinase K for 24 h. After that, DNA was extracted by using distilled water as extraction buffer. PCR amplification was carried out with universal primers spanning the internal transcribed spacer (ITS) region to distinguish species. When using the existing DNA detection method, quantification results showed that 42.86% of the deposited DNA was extracted. Whereas the modified DNA extraction method was increased to 100%. When PCR products test the direct sequencing using the ITS rDNA primers, it was also identified as M. javanica, M. incognita, and M. hispanica. Based on the studies conducted, the application of this modified method would be useful and efficient on plant parasitic nematode molecular assay.