To explore a simple and feasible method for drug injection into zebrafish gonad,the integrated surface projection of the sex gonads was firstly figured out by dissection, precise measurements and calculation of ten adult zebrafish. Injections of the Alcian blue dye were tried to determine the in vitro injection path and injection area. Then dextran texas red and sox9a-myc overexpressed plasmid gene with liposome transfection reagent mixture were injected into the gonads. 48 h later,dextran texas red distribution was examined under fluorescence microscopy and Sox9a-myc expression was determined by using Western blot. Based on the relative size and location of the sex gonads, injection site was determined upward about 0.1 cm from the intersect between pectoral fin upper horizontal and the front end vertical line of ventral fin. 48 h after injection, Alcian blue and dextran texas red fluorescence were observed in the gonad and surrounding organ. Meanwhile overexpression of Sox9a-myc was detected in ovary using Western blotting analysis. These results suggest that our established gonadal injection method is effective and feasible, which could facilitate the in vivo study of zebrafish sex gene regulation via gonadal drug administration.