Alpinia galanga oil (AGO) has an anesthetic activity but its water insoluble property limits its clinical applications. The aim of the present study was to develop a self-nanoemulsifying drug delivery system of AGO (SNEDDS-AGO) to avoid the use of organic solvent and investigate AGO transportation pathway and anesthetic activity. Three optimized formulations from a contour plots of droplet size; SNEDDS-AGO-1, SNEDDS-AGO-2, and SNEDDS-AGO-3, composed of AGO, Miglyol 812, Cremophor RH 40, Capmul MCM EP, and ethanol at the ratios of 40:10:35:10:5, 40:20:15:20:5, and 60:10:15:10:5, respectively were selected as they possessed different droplet size of 62 ± 0.5, 107 ± 2.8, and 207 ± 4.3 nm, respectively. It was found that the droplet size played an important role in fish anesthesia. SNEDDS-AGO-3 showed the longest anesthetic induction time (270 sec) (p < 0.03). Transportation pathway and skin permeation of SNEDDS-AGO-2 were investigated using nile red labelled AGO and detected by fluorescence microscope. AGO was found mostly in brain, gills, and skin suggesting that the transportation pathway of AGO in zebrafish is passing through the gills and skin to the brain. SNEDDS-AGO formulations showed significantly higher permeation through the skin than AGO ethanolic solution. In conclusion, SNEDDS is a promising delivery system of AGO.