AbstractBinderless fiberboards from enzymatic hydrolysis lignin (EHL) and cotton stalk fibers were prepared under various manufacturing conditions, and their physico-mechanical properties were evaluated. Full factorial experimental design was used to assess the effect of fiber moisture content and pressing temperature on boards’ properties. In addition, differential scanning calorimetry (DSC) was used to obtain the glass transition temperature (Tg) of EHL. We found that both fiber moisture content and pressing temperature had significant effects on binderless fiberboards’ properties. High fiber moisture content and pressing temperature are suggested to contribute to the self-bonding improvement among fibers with lignin-rich surface mainly by thermal softening enzymatic hydrolysis lignin. In this experiment, the optimized pressing temperature applied in binderless fiberboard production should be as high as 190°C in accordance with the EHL Tg value of 189.4°C, and the fiber moisture content should be limited to less than 20% with a higher board density of 950 kg/m3 to avoid the delamination of boards during hot pressing.