Biochar application to amend acidified tobacco-soils can enhance tobacco quality and reduce nitrous oxide (N₂O) emissions. Microplastics from agricultural mulch are commonly found in cash-crop farmland soils and, together with biochar, affect soil N₂O emissions. In this study, we applied three types of microplastics (polyethylene, PE; polylactic acid, PLA; polybutylene adipate terephthalate, PBAT) and rice biochar alone or in combination to acidified tobacco planting soil in central China to investigate their effects on soil N₂O emissions, soil chemical properties, nitrogen-cycle-related functional genes, and microbial functional diversity during a 35-day laboratory incubation period. Significant increases in N₂O emissions were observed with PE and PLA, which raised emissions by 15.96 % and 21.52 %, respectively. Additionally, different microplastics affected soil N₂O emissions through distinct regulatory pathways. Co-application of microplastics and biochar suppressed N₂O emissions compared to microplastics alone. Biochar mitigates N₂O emissions mainly by increasing the abundance of the nosZ gene. It can remediate soil contaminated by microplastics and reduce their negative impacts on the soil environment. This study provides deeper insight into the effects of microplastics on soil nitrogen cycling and biochar-mitigated remediation of microplastic-contaminated soil.