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The Achilles tendon plays an important role in our daily activities and studies have shown that tendon properties, such as morphology and stiffness, are related to aging, activity levels and genders. A better understanding of its morphology and mechanical properties will provide guidance to health professionals on intervention recommendation. However, the majority of the existing studies investigated muscle tendon in a single plane and there is a lack of consistent method for 3D anatomical investigation. The objective of this study is to setup a 3D freehand ultrasound imaging system and investigate in vivo 3D Achilles tendon structure in anatomical reference frame. A GE ultrasound machine with motion sensor was used to obtain 3D structure of the Achilles tendon. The ultrasound images were segmented and the digitized points were transformed into the anatomical reference frame. The point cloud data were further meshed using triangulation and smoothed using NURBS. The 3D surface was sliced with horizontal planes to obtain the tendon girth and cross-sectional area as a function of tendon length. The results agree well with previous studies and demonstrate the feasibility of applying this system in the clinical sites.