Human cytochrome P450 3A4 (CYP3A4) is the most abundant phase I drug-metabolizing enzyme in the liver, and approximately 50% of drugs on the market are metabolized by CYP3A4. Therefore, many in vitro studies relied on recombinant CYP3A4 as screening tool to evaluate potential drug-drug interactions (DDIs) in vivo. However, limited information regarding recombinant CYP3A4 with high catalytic activity is available. So, the present study aimed to obtain recombinant CYP3A4 with high catalytic activity and to characterize its functions in vitro. To enhance the catalytic activities of heterologously expressed CYP3A4, the enzyme was fused to cytochrome b5 (b5) tail-to-head, and the fused enzyme was inserted together with NADPH–P450 reductase (POR) into a single plasmid to achieve a simultaneous expression in sf9 cells. Here, substrate binding affinities, enzymatic activities and applications in in vitro DDIs of the fused enzyme were investigated. The dissociation constant Kd of POR-cyt b5CYP3A4 was 8.3 ± 0.87 μmol/L, the Clint (Clint=Vmax/Km) was 8.57 mL/min/g protein for POR-cyt b5CYP3A4 in the metabolism of testosterone and 150.3 mL/min/g protein for midazolam. In addition, the inhibitory constant Ki of ketoconazole on testosterone metabolism was 0.013 ± 0.0038 μmol/L. The present results suggested significantly increased substrate binding affinity and enzymatic activity for the fused enzyme. Thus, the construct could be helpful for studying drug metabolisms and DDIs investigation associated with CYP3A4 in vitro. In addition, simultaneous expression of the fused enzyme and POR could provide more reproducible results based on a more stable molar ratio of CYP3A4/POR/b5.