Focused ultrasound (FUS) lacks reliable real-time image guidance, which hinders the development of non-invasive ultrasound treatment in many important clinical applications. A dual-mode ultrasound array, capable of both imaging and therapy offers a new and reliable strategy for image-guided ultrasound therapy applications. The strategy has the advantages of real-time use, low cost, portability and inherent registration between imaging and therapeutic coordinate systems. In this work, a dual-mode two-dimensional (2D) matrix array with 1 MHz center frequency and 256 elements for ultrasound image-guided non-invasive therapy is reported. The array can provide three-dimensional (3D) volumetric ultrasound imaging and 3D focus control. Ultrasound imaging and therapeutic applications for the brain of small animals demonstrated the multi-functional capability of the dual-mode 2D matrix array. A method of rat brain positioning based on ultrasound imaging was proposed and verified. Transcranial ultrasound image-guided blood–brain barrier (BBB) opening of multiple-targets was achieved in vivo, using the proposed dual-mode 2D array. The obtained results indicate that the dual-mode 2D matrix array is a promising method for practical use in ultrasound image-guided non-invasive therapy applications.