In this study, an innovative approach was proposed based on the implement of biochar derived from residue cornstalk left after anaerobic bio-hydrogen production (RCA-biochar) to improve direct bio-hydrogen production from anaerobic fermentation of cornstalk. The bio-hydrogen production potential and maximum bio-hydrogen production rate increased from 156.2 to 286.1 mL H₂/g substrate and 3.5 to 5.7 mL H₂/g substrate/h, respectively, following the added RCA-biochar increased from 2.5 to 15.0 g/L. Cornstalk chemical component analysis showed the cellulose and hemicellulose content decreased by 17.9–33.7% and 14.4–25.2%, and lignin content increased by 20.3–42.8%, respectively, after 96 h anaerobic fermentation with RCA-biochar 2.5–15.0 g/L. Further analyses revealed that RCA-biochar not only provided more specific surface area for hydrogen-producing bacteria attachment, but also promoted the cellulolytic enzyme activity, thereby resulted in increased substrate conversion to bio-hydrogen. The findings obtained in this study may provide supports for effective and sustainable lignocellulosic bio-hydrogen production in the future.