The accumulation of microplastics in agricultural soil brings unexpected adverse effects on crop growth and soil quality, which is threatening the sustainability of agriculture. Biochar is an emerging soil amendment material of interest as it can remediate soil pollutants. However, the mechanisms underlying biochar alleviated the toxic effects of microplastics in crops and soil were largely unknown. Using a common economic crop, peanut as targeted species, the present study evaluated the plant physiologica and molecular response and rhizosphere microbiome when facing microplastic contamination and biochar amendment. Transcriptome and microbiome analyses were conducted on peanut root and rhizosphere soil treated with CK (no microplastic and no biochar addition), MP (1.5% polystyrene microplastic addition) and MB (1.5% polystyrene microplastic+2% peanut shell biochar addition). The results indicated that microplastics had inhibitory effects on plant root development and rhizosphere bacterial diversity and function. However, biochar application could significantly promote the expressions of key genes associated with antioxidant activities, lignin synthesis, nitrogen transport and energy metabolism to alleviate the reactive oxygen species stress, root structure damage, nutrient transport limitation, and energy metabolism inhibition induced by microplastic contamination on the root. In addition, the peanut rhizosphere microbiome results showed that biochar application could restore the diversity and richness of microbial communities inhibited by microplastic contamination and promote nutrient availability of rhizosphere soil by regulating the abundance of nitrogen cycling-related and organic matter decomposition-related microbial communities. Consequently, the application of biochar could enhance root development by promoting oxidative stress resistance, nitrogen transport and energy metabolism and benefit the rhizosphere microecological environment for root development, thereby improved the plant-soil system health of microplastic-contaminated agroecosystem.