This research focused on ultrasound-enhanced bio-activity of anaerobic sludge. Low energy ultrasound irradiation can increase the bio-activity of anaerobic sludge. Ultrasonic parameter, characteristics of anaerobic sludge and experimental conditions are important parameters which affect the enhancement effect on anaerobic sludge. In order to assess the effects of characteristics of anaerobic sludge and experimental conditions on ultrasonic irradiation of anaerobic sludge, experiments with different characteristics of anaerobic sludge were carried out and analyzed with the content of coenzyme F420 and dehydrogenase activity (DHA). The results showed that anaerobic sludge bio-activity was impacted by the initial temperature, initial chemical oxygen demand (COD), sludge concentration, and stirring during the ultrasonic process. Optimal performance was achieved when sound frequency, power density, and ultrasonic irradiation period was 20 kHz, 0.1 W/mL, and 10 min, respectively, under which the wastewater COD removal efficiency was increased by 12.9 percentage points. The results indicated that low temperature could affect the anaerobic sludge irradiation effect, while intermittent stirring could enhance the bio-activity of anaerobic sludge irradiation effect and low substrate concentration improved anaerobic sludge activity by ultrasound.