Aptamer guided nanomedicine shows great promise in targeted cancer therapies. However the loss of targeting capacity during in vivo or clinical trials has largely hindered its popularity and there are no systematic studies to elucidate the causes. Herein, we investigated such loss of targeting capacity by examining how the physiological milieu affected targeting effect. Aptamer functionalized gold nanoparticle (AuNP) was chosen as the model and exposed to human blood serum that is used to mimic physiological milieu. Dynamic light scattering (DLS), flow cytometry and label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) were employed to determine variations of NPs' surface chemistry and biological identities changes after serum exposure. Results showed that the targeting ability loss was caused by protein corona blocking, replacement and enzymatic cleavage of surface aptamer targeting ligands. Noteworthy, the aggregation issue is critical for the smaller NPs. Analysis of the protein corona profile indicated the accumulation of immune-related proteins on the surface of aptamer-conjugated NPs, which could induce immune response, resulting in rapid clearance of NPs.