Fish are frequently anesthetized to reduce stress and injury during various procedures, particularly surgery. Nonetheless, the majority of chemical fish anesthetics have some negative effects on both fish and humans. 2-Phenoxyethanol (2-PE), for example, which is normally used as an anesthetic for ornamental fish, can cause neuropsychological syndromes in humans. To reduce toxicity, some herbal essential oils have been applied as an anesthetic to fish. However, essential oils must be dissolved in ethanol, which has side effects such as disrupting gas exchange in fish. This study aimed to develop mugwort (Artemisia vulgaris) oil as a nanoemulsion without ethanol and determine their optimal concentrations to use as an anesthetic for surgery on koi carp (Cyprinus carpio). Gas chromatography-mass spectrometry (GC/MS) was used to examine the chemical compositions of mugwort oil. The droplet size of mugwort oil nanoemulsion was measured by the dynamic light scattering method. The results revealed that mugwort oil consisted of three major active compounds which were b-pinene (11.7%), 1,8-cineole (10.34%), and a-pinene (8.29%). The average diameter of mugwort oil nanoemulsion droplets was 22.08 ± 0.70 nm. The lowest effective concentrations were 0.6 ml/L for mugwort oil and 12.5 ml/L for mugwort oil nanoemulsion.