Nicotinamide mononucleotide (NMN) has important biological activities such as anti-cancer and anti-aging. The content of NMN in plants is low, and blocking the degradation pathway is an effective way to increase the NMN content. Research on NMN degradation pathways in plants is still unclear. In this study, eight 5′-nucleotidase candidate genes were cloned from Brassica oleraceae var. acephala, the biological information of the encoded products were analyzed and the proteins were purified using the Escherichia coli expression system. Phylogenetic analysis shows that 5′-nucleotidase is conserved in plants, suggesting that it may play an important role in regulating nucleotide metabolism. In vitro enzymatic reactions were carried out to study the catalytic properties of these 5′-nucleotidases. The results showed that 5′-nucleotidase BolN2, BolN5-X1, and BolN6 from B. oleraceae var. acephala can catalyze the degradation of NMN to generate nicotinamide riboside. In addition, BolN2, BolN5, and BolN6 can also catalyze the hydrolysis of nicotinic acid mononucleotide, purine and pyrimidine nucleotides, with a wide range of substrate adaptability. This study reveals the catalytic properties of 5′-nucleotidase in B. oleraceae var. acephala, laying the foundation for exploring the degradation pathway of NMN in B. oleraceae var. acephala and creating new germplasm with high NMN content.