Sonothrombolysis using diagnostic ultrasound (US) in combination with microbubble (MB) contrast agents is an attractive trial. Superheated perfluorocarbon nanodroplet (SPN), which can turn into MBs upon US trigger, may have advantages in sonothrombolysis. As a preliminary investigation of SPN-assisted sonothrombolysis, we performed a safety evaluation in vivo . Twenty male rabbits (2.59 ± 0.14 kg) were assigned to three groups: the Control group ( n = 6), 2.2 mL/kg of physiological saline intravascular (i.v.) injection into auricular vein; the PL group ( n = 8), 25 mg/kg of phospholipid-coated SPN i.v.; and the AA group ( n = 6), 25 mg/kg of SPN coated with poly aspartic acid derivative i.v. Rectal temperatures were maintained at 39.08 ± 0.98 °C. Neurological evaluation and biochemical blood examinations were performed at pre-injection, 1, 4, and 7 days after injection. Organ samples including heart, lungs, liver, spleen and kidneys were harvested after euthanasia. Within an hour after administration of SPNs, both the PL and AA groups showed a reversible change in respiration. One animal in the AA showed transient nystagmus about 20 min after administration; however, there was no pathological damage. One animal in the PL died 2 days after. No histological damage was found in any organ sample from any of the animals. Moreover, no significant differences were found in the biochemical blood examination between the PL, AA, and Control groups. No neurological damage or histological change was found with two SPNs. We will further investigate the SPN-assisted sonothrombolysis based on the 500-kHz US exposure with bubble liposome acceleration of rt-PA efficacy.