Based on the nucleotide sequence of phytoene desaturase gene NtPDS of Nicotiana benthamiana published on NCBI, specific primers were designed and synthesized. With the total RNA from leaves of Nicotiana tabacum cv. Hongda used as the template, the NtPDS gene was obtained by PCR. Sequence analysis showed that the gene contained a full coding region of 1,749 bp encoding 582 amino acid residues with a the molecular mass of 65.04 kDa and the isoelectric point of 7.53. The fragments of the NtPDS gene were cloned into the prokaryotic expression vector pET-32a and the fusion expression vector pET-32a-NtPDS was constructed, which was then transformed into Escherichia coli BL21(DE3). The fusion protein NtPDS in the form of soluble fraction was effectively expressed under 1 mmol/L IPTG concentrations at 37°C for 4 hours. And then this result was proved by Western blotting. The semi-quantitative PCR results revealed that the transcript of NtPDS gene was detectable in leaves, flowers and stems, but no such transcript detected in the roots. It laid a foundation for the further study on the enzyme activity of NtPDS and its functions.