To reveal the biological function of fructokinase (OsFRK) family genes in rice, two knock-out mutants for identified OsFRK family genes were constructed using CRISPR/Cas9 technology. The results showed that 28 OsFRK1 and 14 OsFRK2 T0 transgenic plants were successfully constructed. The transgenic positive rate, mutation rate and homozygous mutation rate of OsFRK1 is 100%, 46.43% and 10.71%, while OsFRK2 is 92.86%, 92.86% and 21.43%. In addition, the functional domains of targeted proteins of the T0 knock-out homozygous mutants were damaged in the mutatnts.