Biochar, which has emerged as an important form of the transformation and final disposal of biomass, can be used directly in soil or in seedling nurseries. In this study, the use of biochar of different particle sizes and percentages was evaluated in replacement to a conventional substrate used in the production of sugarcane seedlings. To this end, an experiment was carried out based on a completely randomized design, with a 5 × 4 factorial scheme, consisting of five different percentages of biochar (with 0, 25, 50, 75, and 100% v/v substitution of the conventional substrate) and four particle sizes (<1, 2, 4, and 9 mm), with nine repetitions. As seedling growth variables, the average sprouting time, sprouting speed index, plant height, leaf number, leaf length, and width + 2, as well as the dry mass of the aerial parts and roots were evaluated. Irrespective of the percentage of commercial substrate replaced with biochar, sprouting time was found to be shorter when 6-mm-diameter biochar particles were used. With respect to the sprouting speed index, it was found that regardless of particle size, the highest value occurred when biochar was used to replace 42% of the commercial substrate. The substitution of the commercial substrate with biochar had the effect of reducing the growth of sugarcane seedlings.