In response to the low removal capacity and poor tolerance of fungi to diesel-contaminated soil, a novel immobilization system using biochar to enhance composite fungi was proposed. Rice husk biochar (RHB) and sodium alginate (SA) were used as immobilization matrices for composite fungi, and the adsorption system (CFI-RHB) and the encapsulation system (CFI-RHB/SA) were obtained. CFI-RHB/SA exhibited the highest diesel removal efficiency (64.10%) in high diesel-contaminated soil over a 60-day remediation period compared to the free composite fungi (42.70%) and CFI-RHB (49.13%). SEM demonstrated that the composite fungi were confirmed to be well attached to the matrix in both CFI-RHB and CFI-RHB/SA. FTIR analysis revealed the appearance of new vibration peaks in diesel-contaminated soil remediated by immobilized microorganisms, demonstrating changes in the molecular structure of diesel before and after degradation. Furthermore, CFI-RHB/SA maintains a stable removal efficiency (>60%) in higher concentrations of diesel-contaminated soil. High-throughput sequencing results indicated that Fusarium and Penicillium played a key role in the removal of diesel contaminants. Meanwhile, both dominant genera were negatively correlated with diesel concentration. The addition of exogenous fungi stimulated the enrichment of functional fungi. The insights gained from experiment and theory help to provide a new understanding of immobilization techniques of composite fungi and the evolution of fungal community structure.