Drug metabolizing enzyme CYP3A65 is the only member of Danio rerio CYP3A subfamily. Our study was conducted to select appropriate D. rerio developmental stage, by investigating the expression of CYP3A65 and its its upstream nuclear receptor (PXR and AHR2) during developmental stages.The zebrafish in different growth stages were raised, and the whole fish tissues, liver tissues and intestinal tissues were taken. The relative expression levels of CYP3A65, PXR and AHR2 were measured, and the correlation between nuclear receptors and metabolic enzyme mRNA was compared to confirm the regulating effect of nuclear receptors.In whole fish, liver and intestine tissues, the mRNA levels of CYP3A65, PXR and AHR2 were the highest in 144 hpf larvae and 75 dpf juvenile D. rerio. The change trends of metabolic enzyme and nuclear receptors gene expression were consistent, and affected by the proportion of liver and intestine relative to whole fish. The 144 hpf and 75 dpf D. rerio may be in the juvenile stage and throughout the growth phase, and are most suitable for CYP3A65 metabolism studies. There were significant correlations between CYP3A65 and PXR、CYP3A65 and AHR2 mRNA expression, and correlation between CYP3A65 and AHR2 was better. In our later work, the enzyme activity and protein expression will be further investigated during D. rerio development, which would provide more information for the model organism D. rerio.