Ferredoxin (Fdx) is regarded as the main electron carrier in biological electron transfer and acts as an electron donor in metabolic pathways of many organisms. Here, we screened a self-sufficient P450-derived reductase PRF with promising NADPH reduction activity and 9OHAD production yield and proved the importance of [2Fe-2S] clusters of Fdx-containing oxidoreductase in transferring electrons in steroidal conversion. The truncated Fdx domain in all oxidoreductases, together with mutagenesis data, further elucidated the indispensable role of [2Fe-2S] clusters in the electron transfer process. By adding the independent plant-type Fdx to the reaction system, the AD conversion rate have been significantly improved. A novel efficient electron transfer pathway of PRF+Fdx+KshA in the reaction system rather than KshAB complex system was proposed based on analysis of protein-protein interactions and redox potential measurement. Adding free Fdx created a new conduit for electrons to travel from reductase to oxygenase. This electron transfer pathway provides new insight for the development of efficient exogenous Fdx as an electron carrier.