Plant fructokinase (FRK) specifically catalyzes fructose phosphorylation into glycolysis pathway, which is a key enzyme for fructose metabolism and soluble sugar accumulation. This study elucidated the expression pattern and enzyme characteristics of red pitaya HpFRK2 gene, which would provide a theoretical basis for understanding the molecular mechanism of fruit soluble sugar accumulation and improving fruit quality. HpFRK2 was cloned from red pitaya ‘Zihonglong’, and its expression pattern was analyzed by real-time fluorescence quantitative PCR. Subcellular localization was performed by transiently expressing in tobacco mesophyll cells, and the recombinant protein was obtained by prokaryotic expression and its enzyme activity was detected. The open reading frame (ORF) was 1026 bp, encoding 341 amino acids. The relative molecular weight of HpFRK2 was 36.95 kDa, and the theoretical isoelectric point was 5.94. Phylogenetic analysis showed that HpFRK2 was closely related to cassava MeFRK2, tomato SlFRK1, apple MdFRK1 and Arabidopsis AtFRK1. HpFRK2 contained a conserved domain of the phosphofructokinase type B (pfkB) family. The expression level of HpFRK2 was the highest at 20 days of fruit development, gradually decreased with fruit development, and the expression level was the lowest at 30 days. In addition, the expression of HpFRK2 in stems was significantly lower than that in fruits. Subcellular localization results showed that HpFRK2 was mainly located in the cytoplasm. A prokaryotic expression vector was constructed and expressed in Escherichia coli, and recombinant protein was successfully induced. The results of enzyme activity characteristics showed that HpFRK2 specifically catalyzed fructose phosphorylation, fructose did not have the substrate inhibition on its enzyme activity, and the Km value for fructose phosphorylation was 1.84 mmol/L. The results showed that HpFRK2 was located in the cytoplasm, specifically catalyzed fructose phosphorylation, was mainly expressed during the veraison period (20-23 d after flowering) of red pitaya fruit, and may negatively regulate fructose accumulation in fruit.