Biochar has been used to strengthen anaerobic digestion (AD) for several decades. However, the ability of low-cost biochar from agricultural waste, such as corn straw to enhance pilot-scale continuous-flow AD, has not been widely applied and systematically investigated. In this study, AD fed with corn straw was explored in 16 L continuous stirred-tank reactors with the addition of corn straw and coconut shell biochar. In addition, the effects of both substrate concentration and hydraulic retention time (HRT) on the enhanced AD system were explored. As a result, an appropriate increase in substrate concentration could improve the daily biogas production rate, and the highest value was 1.41 L L⁻¹ d⁻¹ at a substrate concentration of 12% when 4.0% of corn straw biochar was used. In addition, the HRT had a significant effect on the microbial characteristics. Methanosarcina performed better at relieving ammonia nitrogen inhibition. There was a positive correlation between p-Bacteroidetes and p-Firmicutes in bacteria and between p-Firmicutes and p-Euryarchaeota in archaea. This study provides crucial insights into the application of low-cost biochar and the research on the enhancement mechanism of AD in the future.