Abstract: The increased demand of diesel fuel enhanced the risk of diesel leaks causing deleterious effects on the aquatic environments. However, attempts to establish useful tools that use environmental DNA (eDNA) to detect genotoxicity by water environment contaminants have received little attention. The present study used RAPD (Random Amplified Polymorphic DNA) technology to detect the genotoxicity of soluble fraction of 0^# diesel on the zebrafish (Danio rerio) using zebrafish genomic DNA and water environment DNA (eDNA) as templates. The results showed no significant change in the RAPD fingerprint of both genomic DNA and eDNA within 96h under control condition. When zebrafish were exposed to different concentration diesel with the prolonged exposure time (0, 24h, 48h, 72h, 96h), the polymorphic sites and template stability of genomic DNA and eDNA decreased. The increased diesel concentration (15%, 50%, 100%) reduced the polymorphic sites and the template stability of genomic DNA and eDNA. These results indicated that 0^# diesel has a time- and concentration-dependent effects on the genotoxicity of zebrafish genomic DNA and eDNA. Moreover, the pattern of RAPD amplification fingerprint of genomic DNA was consistent with that of eDNA RAPD amplification fingerprint. Our research provides new research clues and technical means for detecting the genotoxicity of diesel oil on aquatic organisms by measuring the eDNA damage in water environment using RAPD technique.