This paper investigates the tool-ply friction behavior of woven jute fabric reinforced poly-lactic acid (jute/PLA) biocomposite in thermoforming. A home-made pull-through friction testing device was developed to characterize the tool-ply friction behavior of the jute/PLA prepreg at elevated temperature. The effects of alkali treatment, fiber orientation, normal force and slipping velocity were studied. The results suggested a strong relationship between the tool-ply friction behavior and the woven and fiber structures. In addition, a quantitative definition of the tool-ply friction behavior was achieved by introducing the characteristic stress and friction coefficient. These findings indicated a hydrodynamic lubrication of tool-ply friction. This work provides a beneficial complement to the understanding of the thermoforming process of jute/PLA biocomposites.