The invention discloses a preparation method of double-targeting cationic ultrasound microbubbles carrying cell-penetrating peptide iRGD and belongs to the field of fundamental research. The preparation method uses materials, including DSPC, DSPE-PEG2000-maleimide, DSPE-PEG2000-Biotin, iRGD peptide, CCR2 antibody, stearic acid, branched-chain polyetherimide-600, N, N'-carbonyldiimidazole, and avidin; by bonding integrin Alpha v Beta3 targeting cell-penetrating peptide iRGD and the CCR2 antibody to surfaces of the microbubbles, double-targeting cationic ultrasonic contrast agent is constructed and has the double functions of carrying plasmid DNA and targeting tumor cells; combining ultrasound-mediated biological effect and iRGD cell-penetrating action, the agent is capable of promoting entry of genes into the tumor cells and improving gene transfection efficiency and is expected to be developed and applied to visual ultrasound-controlled-release genetic treatment of tumors.