Intense droughts and extreme precipitation events are likely to occur more frequently with global climate change. These drying-rewetting (DW) cycles affect the soil carbon (C) cycle. Biochar addition are reported to affect SOC mineralization and soil organic carbon (SOC) storage. However, the effects of biochar application on SOC mineralization during DW cycles are poorly understood. Two wheat straw (WS25) biochar produced at 300 °C (WS300) and 600 °C (WS600) were used to explore the effects of biochar on SOC mineralization under artificial DW cycles as compared to constant moisture (CM). It was found that biochar had different effects on SOC mineralization depending on biochar type or drying/rewetting period of DW cycles. Just like CK and WS25, WS600 application decreased SOC mineralization under DW cycles compared to CM. To some extent, SOC mineralization during DW cycles was similar to CM for WS300. The results suggested that WS300 addition diminished the reducing effect of DW cycle on SOC mineralization. In addition, biochar exhibited different effects on SOC mineralization depending on the drying and rewetting period under DW cycles. Biochar (WS300) addition during the drying period had less effect on SOC mineralization but increased the flush effect of SOC mineralization during the rewetting period. In conclusions, biochar application significantly affect SOC mineralization following DW cycles.