The invention discloses a reagent kit and detection method for detecting C/EBPalpha in cells through an amino-modified gold nanocarrier and application of the reagent kit. The reagent kit contains amino-modified gold nanocarriers, membrane penetrating proteins and a double-stranded DNA reagent; and the DNA reagent contains C/EBPalpha binding sites and fluorescent mark. Because positively charged aminated AuNPs can absorb the negatively charged double-stranded DNA containing the C/EBPalpha specific binding sites, fluorescent molecules carried by the AuNPs can be quenched due to the unique optical effect of the AuNPs. When C/EBPalpha exists, the C/EBPalpha can be specifically bound to the double-stranded DNA, thus the adsorption of dsDNA by AuNPs is hindered, and the recovery of fluorescenceintensity is caused. According to reagent kit and detection method for detecting the C/EBPalpha in cells through the amino-modified gold nanocarrier and the application of the reagent kit, detectingthe C/EBPalpha as low as 29 pM without the participation of any signal amplification or other synthetic complexes can be realized.