TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. The CEL I enzyme that specifically cleaves the mismatch in DNA double strands is an important component of the TILLING experiment platform. Due to the expensive price of commercial CEL I, it was not conducive to large-scale TILLING experiments. Referring to published literature, the active crude extract of CEL I enzyme was obtained from celery growing in the Guiyang, Guizhou province. In this study, an effective CEL I digestion system was established as follows: The CEL I enzyme effectively cut the mismatch DNA in the 15µL digestion reaction solution including 8µL PCR product, 4.5µL ddH2O, 1.5µL 10× cleavage buffer (pH 7.5, 500mmol/ L KCL, 100mmol / L Tris-Cl, 15mmol / L MgCl2) and 1µL of the 20-fold dilution of CEL I crude extraction after 30-min incubation at 42°C. We hope that the use of this reverse genetics resource will provide novel allelic diversity for tobacco functional genomics as a model organism.