The prenylation of flavonoids is a main type of structural modification and can endow flavonoids with greater bioactivity and bioavailability. A soluble prenyltransferase (NgFPT) gene from Nocardiopsis gilva was cloned, expressed and characterized in Escherichia coli. The optimal activity of NgFPT was at pH 7.5 and 30 °C. The activity of NgFPT was significantly enhanced by Ca2+, Al3+, and DMSO. NgFPT showed high selectivity to prenylate flavanones at 3′-C to generate 3′-C-prenyl-flavanones. The Kcat and Km of recombinant NgFPT for naringenin were 0.001 s−1 and 0.045 mM, respectively. Then, recombinant strains were reconstructed by introducing NgFPT gene and the isopentenol utilization pathway. Escherichia coli hosts and fusion tags were screened to improve the yield of 3′-C-prenyl-naringenin in vivo, resulting in maximal 3′-C-prenyl-naringenin production at 3.5 mg/L. By optimizing biotransformation conditions and adopting the resting cell bioconversion, maximum 3′-C-prenyl-naringenin production reached 10.3 mg/L with a specific productivity of 0.21 mg/L/h after 48 h incubation. Thus, the article provides a regiospecific soluble prenyltransferase and a method for the production of 3′-C-prenyl-naringenin by metabolic engineering.