Biochar incorporation into soil has been found to benefit crop yield. However, the effects of biochar application on crop root growth and morphology and root endophytic fungi in different crop growth stages remain obscure. Here, a field experiment was established with various biochar rates [0 (control), 4.5 t ha−1 (BC4.5), 9 t ha−1 (BC9)] to examine the effect of biochar application on root development and community structure of root endophytic fungi in a wheat-maize rotation system under semi-arid climate on alluvial soil. The results showed that in BC9 the total root length of maize and shoot nutrient accumulation in the flowering stage were comparable with the control and BC4.5. However, in the grain-filling stage, the root length remained high in BC9, but it was reduced by 21–34% in the control compared with the flowering period, suggesting biochar delayed root senescence. Similarly, the higher specific root length was found in BC9 compared with the control at the grain-filling stage. Biochar also altered the relative abundance and diversity of root endophytic fungi. Ascomycetes and Basidiomycetes dominated the root fungal community, accounting for 57–85% of the total endophytes. Biochar changed the core species in the community structure from Basidiomycetes to Ascomycetes and promoted shoot nutrient accumulation and final grain yield of wheat and maize. Thus, biochar maybe increases crop yield by increasing root length, delaying root senescence, and altering the community structure of root endophytic fungi.