Interferon-γ (IFN-γ) is an important cytokine, which can be used as a biomarker for disease prevention, diagnosis, and prognosis detection in clinics. To achieve highly sensitive detection of IFN-γ, an aptasensor based on hybrid structure of antibodies and aptamer was developed, which involves two probes. Firstly, 4-Mercaptobenzoic acid (4-MBA) was embedded into Au-Ag Alloy nanoparticles, and then coupled with the aptamer as detection probes. Secondly, magnetic beads modified by antibodies were used as capture probes. When IFN-γ is present, IFN-γ can be captured by both aptamers and antibodies, forming a stable sandwich structure, and obtaining strong Raman signals. On the contrary, when there is not IFN-γ, weak Raman signals are obtained. We optimized the relevant parameters such as volume of 4-MBA and antibody, concentration of aptamer. The experimental results show that this method has high sensitivity and repeatability, with a limit of detection (LOD) of 0.25 pg/mL in PBS. Due to the high recovery rate of this method in serum ranging from 98.7% to 108%, we believe that this aptasensor can achieve the fast and accurate detection of interferon-γ in complex serum environments to meet clinical needs.