A fluorophore labeled ATP aptamer probe was combined with probe technique-based generalized multivariate standard addition strategy (GSAMprobe) for accurately detecting the content of ATP in complex biological samples. The experimental results showed the ratio of fluorescence signal intensity (F/F0) of the reaction system had a good linear relationship with the concentration of ATP ranged from 5~65 μmol/L, and the linear correlation coefficient R2 was 0.987. Cell lysate and human plasma were selected as the complex sample simulated system to detect the concentration of ATP. Experimental results demonstrated that one standard addition sample was enough for the proposed strategy to provide satisfactory quantitative results compared with classical univariate method and PLS method. This strategy is expected to be applied to the rapid and accurate quantitative analysis of other small-volume biological samples or some precious samples.