Abstract: The Pictet-Spenglerase（P-S enzyme） is essential for the biosynthesis of carboline alkaloids. The P-S enzyme catalyzes the formation of the β-carboline skeleton by condensing the tryptophan or tryptamine with the corresponding aldehyde or ketone substrate. Nocardiopsis flavescens CGMCC 4.5723 produces a β-carboline alkaloid called Marinacarboline A, suggesting that a new P-S enzyme exists in the Marinacarboline A biosynthetic pathway. In this study, the gene sequence encoding the known P-S enzyme of McbB/NscbB was used as molecular marker, the coding gene of P-S enzyme in the strain of N. flavescens CGMCC 4.5723 was located, and the enzyme was named NfmcbB. Further characterization of NfmcbB confirmed that NfmcbB can condense the L-tryptophan and methylglyoxal to biosynthesize 1-acetyl-3-carboxyl-β-carboline and 1-acetyl-β-carboline, two precursors of Marinecarboline with β-carboline skeleton. Our study expands the biosynthesis research of β-carboline alkaloid, and further provides a new P-S enzyme candidate for developing biocatalyst in the future.