Highly sensitive detection of proteins is essential to biomedical research as well as clinical diagnosis. However, usual detecting methods are complicated operating, time-costing and extensive label preparing. Here, we combined molecular beacon (MB) track mediated DNA walker and nicking enzyme assisted signal amplification method to develop a simple and ultrasensitive malachite green fluorescence biosensor for specific detection cytokine, interferon-γ (IFN-γ). The association of the IFN-γ with the corresponding aptamers of the dsDNA strands leads to free of DNA walker which trigged the generation of DNA track at the help of Nicking endonuclease (Nb.BbvCI). The released MB track opens MB2 to form MB track/MB2 duplex and the duplex cleaved the MB track with the help of Nb.BbvCI. The DNA track is subsequently released to hybridize with another MB2. And the cleavage also generates the G-rich oligomer, this released G-rich oligomer folds into a G-quadruplex structure and thus allows the formation of a fluorescence transducer in the presence of Malachite green (MG). The formed fluorescence transducer can give a high fluorescence intensity. So, one IFN-γ can initiate the cleavage of numerous MB track and MB2, resulting in the highly sensitive detection of IFN-γ with the detection limit of 7.65 fM. This new methodology can be expected to provide a highly sensitive platform for the amplified analysis of various target molecules.