Chiral triazole pesticides may cause enantioselectively adverse effects to non-target organisms. In this work, we employed zebrafish as an aquatic organism model to explore stereoselective acute toxicity, bioaccumulation, oxidative stress, and thyroid disruption of cis-metconazole enantiomers. The median lethal concentration values of (1S, 5R)-metconazole, (1R, 5S)-metconazole, and the mixture of them against zebrafish were 4.01, 2.61 and 3.17 mg⋅L⁻¹, respectively. (1R, 5S)-Metconazole was preferentially bioaccumulated in zebrafish than (1S, 5R)-metconazole, and the bioconcentration factor of (1R, 5S)-metconazole was 1.28-fold larger than that of (1S, 5R)-metconazole. Then, the activity order of catalase, superoxide dismutase, and glutathione-S transferase enzymes in zebrafish was expressed as (1S, 5R)-metconazole > the mixture > (1R, 5S)-metconazole, while the order of malondialdehyde content in zebrafish was (1R, 5S)-metconazole > the mixture > (1S, 5R)-metconazole. Moreover, cis-metconazole exhibited enantioselective regulation effects on the levels of triiodothyronine and thyroxine in zebrafish, and (1R, 5S)-metconazole possessed stronger thyroid disruption ability to zebrafish than the others. By virtue of molecular docking methodology, the binding affair and docking energy results supported that interactions between (1R, 5S)-metconazole and thyroid hormone receptors were much stronger than those between (1S, 5R)-metconazole and same receptors. This study of enantioselective evaluation of cis-metconazole in zebrafish can provide favorable information for risk assessments of chiral pesticides toward environment and health of aquatic organisms.