Ultrasound is useful in demonstrating bone mineral density of regenerating osseous tissue as well as structural alterations. A proposed ultrasound method, which included ultrasonography and acoustic parameters measurement, was employed to evaluate its efficacy in monitoring the bone callus changes in a rabbit tibial distraction osteogenesis (DO) model. The findings demonstrated that ultrasonographic images depicted characteristic changes of the bone callus, typical of histology findings, during the distraction phase. Follow-up acoustic parameters measurement of the bone callus, including speed of sound, reflection and attenuation, showed significant linear changes over time during the distraction phase. The acoustic parameters obtained during the distraction phase also showed moderate to strong correlation with consolidated bone callus density and micro-architecture measured by micro-computed tomography at the end of the consolidation phase. The results support the preferred use of ultrasound imaging in the early monitoring of bone callus changes during DO treatment.