Since plug seedling plays a key role in automatic transplanting, this work aimed to explore the effect biochar has on the root growth of plug seedlings. The physicochemical properties tests showed that the addition of biochar in the peats could increase the porosity, pH, and EC values of the substrate, and the substrates treated with 0%, 10%, 20%, and 30% biochar could meet the requirements of seedling raising. The water retention of the substrate was superior with the increase of biochar proportion, and the nitrogen release significantly decreased with the increase of the biochar proportion. Our results demonstrated that the substrate with 10% biochar-treated apparently promoted the growth of seedlings and root systems, even the length of the root-tip cells. However, the substrates with 40% and 50% biochar-treated obviously inhibited the growth of seedlings and root systems. It was noticed that the strength of substrate with appropriate biochar proportion was enhanced, as well. Under the interaction of strong root system and solid substrate, the compressive strength of the substrate with 20% and 10% biochar-treated was much better than others, especially that of 40% and 50% biochar-treated, which efficiently satisfied the requirements of automatic seedling picking. The biochar may have a good application prospect in seedling raising.