Denitrifying Anaerobic Methane Oxidation (DAMO), which utilizes methane for denitrification, offers an efficacious approach for nitrogen removal in wastewater and greenhouse gas mitigation. However, slow microbial growth and low methane mass transfer efficiency limit its practical application. This study demonstrates that straw biochar, particularly when pyrolyzed at 300 °C, enhances DAMO's nitrogen removal performance. Specifically, biochar from cotton (CB300) and maize (MB300) stalks at 300 °C increased the nitrate removal rate by 2.6 and 2.4 times, respectively, compared to the control. Correlation analysis revealed a positive link between nitrate removal rates and oxygen-containing functional groups in biochar, which may facilitate electron transfer. Long-term DAMO reactor operation confirmed significant enhancements in nitrogen removal with 300 °C biochars. CB300 and MB300 biochars increased key functional genes (mcrA and pmoA) and enriched DAMO archaea (ANME-2D). These findings suggest that low-temperature biochar is a cost-effective and sustainable approach to enhance the nitrogen removal performance of DAMO.