The invention provides a fluorescence detection kit for a deletion mutation gene. The kit includes: a PNA capture probe, a DNA probe, a padlock probe, a DNA polymerase, a DNA ligase, a rolling circleamplification primer, and a fluorescence probe. The invention also provides a method of using the kit to perform the fluorescence detection on the deletion mutation gene, wherein the method includes:1) immobilizing the PNA capture probe on the bottom of a pore plate, and hybridizing the probe with a target gene with a buffer solution; 2) hybridizing the target gene immobilized on the bottom of the pore plate with the DNA probe; 3) hybridizing a single chain section on the DNA probe, which is hybridized with the target gene, with the padlock probe, cyclizing the hybridized product with the DNAligase, and performing rolling circle amplification under effect of the rolling circle amplification primer and the DNA polymerase; 4) hybridizing the fluorescence probe with a rolling circle amplification product, quenching background fluorescence, and detecting the change on fluorescence intensity.