In this study, soil samples were obtained from citrus orchard from Huazhong Agricultural University. Using valencene as the only carbon source, the strains with good growth characteristics were firstly screened according to the growth curve measured in the minimum culture medium. And the strains with high conversion amount of nootkatone were screened again. The types and contents of the products produced by valencene biotransformation were measured and analyzed using SPME-GC-MS. Through physiological and biochemical tests and 16SrDNA sequencing, the screened strains were identified as Burkholderia sp., which was named Burkholderia sp. PZQ14. Single-factor test of fermentation medium components was conducted using LB medium as a starting point and nootkatone yield as an index. On this basis, the effect of different concentration of culture medium components on nootkatone yield was investigated by response surface optimization test. The results showed that the optimal medium components were: glucose concentration of 38 g/L, peptone concentration of 25 g/L, and FeSO4 concentration of 0.60 g/L. The content of nootkatone was 328.69 ± 17.88 mg/L and the conversion ratio was 35.73 ± 1.24% when three validation tests were conducted under these conditions. The nootkatone yield increased by 251 mg/L and the conversion ratio increased by 27.30% compared to LB medium.