Herein, a dual-signal ratiometric fluorescence-based p53 gene sensing strategy was developed for the first time. Nitrogen doped graphene quantum dots (NGQDs) were firstly bound with a single-stranded DNA (P1 DNA), which contains berberine aptamer sequence and p53 gene complementary sequence (Cp53 DNA). With the addition of berberine, berberine will bind to berberine aptamer in P1 DNA, and a dual-signal ratiometric fluorescence probe NGQDs/P1 DNA/berberine was established, which displays two fluorescence peaks at 440 nm and 537 nm corresponding to NGQDs and berberine, respectively. P1 DNA can be hydrolyzed by Exonuclease I (Exo I) when target p53 gene was absent, resulting in the release of berberine and the decrease of fluorescence intensity at 537 nm. When target p53 gene was present, Cp53 DNA sequence in P1 DNA can specifically bind to p53 gene and form double-stranded DNA, so the hydrolysis of P1 DNA by Exo I was blocked, and the fluorescence intensity at 537 nm was recovered. The fluorescence of NGQDs at 440 nm does not affected from Exo I and target p53 gene, and was employed as reference. The sensing strategy showed a good linear response to p53 gene in the range of 0.2-30.0 nM with a detection limit (LOD) of 0.06 nM, and it performed well in human serum samples.