Flavin mononucleotide (FMN) plays an important role in living systems. Accurate probing and characterizing of FMN is the key to understanding the contribution of FMN coenzyme in biological processes. We demonstrate a way to get a bright surface-enhanced Raman spectroscopy (SERS) signal while simultaneously quenching the fluorescence background through silver nanorod (AgNR) array substrates fabricated by the oblique angle deposition method. The AgNR array substrates show a good SERS activity with an enhancement factor of ∼4.2  ×  10⁴. Meanwhile, the characteristic peaks of FMN are assigned by the density function theory calculation. An exponential relationship is demonstrated between SERS intensity and FMN concentration in the concentration range of 0.1 to 100  mg  /  L. This SERS-based method enabled the rapid and highly sensitive detection of FMN, holding great promise for expanding SERS application in biochemistry and food safety fields.