Ultrasonic treatment has been shown to have a favorable effect on the regeneration of spent biological activated carbon (BAC) from drinking water treatment plants. In this study, the use of ultrasound as a regeneration method had a significant effect on the recovery of spent BAC after 7.5 years of use; it effectively increased the iodine value from 300 mg/g to 600 mg/g and restored the specific surface area and pore volume of BAC. Ultrasound effectively changed the structure of the biofilm inside and on the surfaces of BAC particles, on the basis of confocal laser scanning microscopy (CLSM) images. The thickness of the surface biofilm attached to BAC reached an “active” level (about 100 μm) at the regeneration frequency of 40 kHz. The dehydrogenase activity significantly improved from 4.50 mg TF/g BAC to 9.13 mg TF/g BAC, and the content of adenosine-triphosphate (ATP) in regenerated BAC was maintained at a high level (2.501 × 10 −6 g ATP/g BAC), thus allowing the development of microbial growth. The production of soluble microbial products (SMPs) from regenerated BAC decreased during the reuse process. The removal efficiency of DOC, COD Mn , NH 4+ and NO 3– control increased by approximately 78%, 71%, 50% and 20%, respectively.