This paper provides a 3D ultrasound navigation system with a blood vessel network (BVN) reconstruction algorithm for microbubble delivery therapy, which manipulates microbubbles in blood vessel by acoustic radiation force of ultrasound, aiming to increase the efficiency of high-intensity focused ultrasound (HIFU) and realize acoustic drug/gene delivery. To apply the technique in vivo, reconstruction of the BVN from 3D volume, such as bifurcation positions and flow direction, and visualization of the relative positions of the BVN and ultrasound field for microbubble manipulation are required. To address these issues, we have developed a 3D ultrasound navigation system to guide microbubbles in blood vessel. The novel navigation system consists of an ultrasound imaging device with a 2D array probe, an optical tracking device, a focused ultrasound transducer, and a Windows workstation with in-house navigation software. The system visualizes three-dimensionally both the BVN and the focal position of the transducer. When a 3D volume is acquired by an imaging device, the volume is automatically sent to the navigation system. Then, the system visualizes the volume and its bifurcations, which are estimated by 3D thinning processing. In this study, we examined the feasibility of the system by evaluating the guidance accuracy and microbubble induction rate. From the results, we confirm that microbubbles can be navigated by the system.