The application of ultrasonic irradiation (40 KHz, 120 W) in the enzymatic extraction of bovine tendon collagen has been investigated. Our results show that using the ultrasonic irradiation increases the yield of collagen up to 124% and significantly shortens the extraction time in comparison with the conventional pepsin isolation method. Such improvements are attributed to the enhancement of the enzyme activity and the dissolution of collagen substrate because the ultrasonic irradiation disperses the pepsin aggregates and opens up the collagen fibrils, thus the enzymatic hydrolysis is facilitated. AFM imaging shows the same fibrillar structure of tendon collagens generated from both the methods. The CD and FT-IR measurements reveal that the triple helix structure of collagen remains intact even after the ultrasonic irradiation. This study shows that the mild ultrasound irradiation can effectively improve the efficiency of pepsin extraction of natural collagen without any compromise of the resultant collagen quality.