NADPH-cytochrome P450 reductase (CPR) is a key enzyme transferring electrons to cytochrome P450. Plants cytochrome P450s play an important role in various metabolisms, biosynthesis, mechanism of hormones and defense mechanisms. In tomatoes (Solanum lycopersicum), two putative CPR genes, SlCPR1 and SlCPR2, were identified. In all the tested tomato tissues, SlCPR2 showed higher expression levels than SlCPR1. SlCPR1 was constitutively expressed, whereas SlCPR2 expression was increased significantly by jasmonic acid treatment. No significant changes were observed with salicylic acid or drought stress treatment. To characterize the enzymatic properties of SlCPRs, the cDNA of SlCPRs were expressed in Escherichia coli without any amino acid modification and purified using ADP-agarose affinity column. Purified SlCPR enzymes were reacted with several protein and chemical substrates. SlCPR2 was more active than SlCPR1. Both SlCPR1 and SlCPR2 exhibited strong activity across a pH range of 6.0 to 9.0, with peak activity at pH 8.0. This study opens possibilities for CPR control, biocatalyst development, and exploring oxidase enzyme functions.