Methylation-mediated pathways play an important role in the progression of cancer. Inhibitors of several key methyltransferases including DNA methyltransferases and histone methyltransferases have proven to be instrumental for both understanding the function of the respective enzymes activites and translational applications in cancer epigenetic therapy. Nicotinamide N-methyltransferase (NNMT) is a major metabolic enzyme involved in epigenetic regulation through catalysis of methyl transfer from the cofactor S-adenosyl-L-methionine, onto nicotinamide and other pyridines, to form S-adenosyl homocysteine and 1-methyl-nicotinamide or the corresponding pyridinium ions. Accumulating evidence infers that NNMT is a novel therapeutic target for a variety of diseases such as cancer, diabetes, obesity, cardiovascular and neurodegenerative diseases. Therefore, there is an urgent need to discover potent and specific inhibitors for NNMT. Herein, we reported the design and synthesis of a fluorescent probe II138, and established a fluorescence polarization (FP)-based competition assay for evaluation of NNMT inhibitors. Importantly, the unique feature of this FP competition assay is its capability to identify inhibitors that interfere with the interaction of the NNMT active site directly or allosterically. In addition, this assay performane is robust with a Z factor of 0.76, and applicable in high-throughput screening for inhibitors for NNMT.