The generation and accumulation of amyloid-beta peptide (Aβ) in amyloid plaques are key characteristics of Alzheimer's disease (AD); thus, specific detection of Aβ is essential for the diagnosis and treatment of AD. Herein, an aptamer-conjugated graphene oxide (Apt-GO) sensor was synthesized by π-π and hydrophobic interactions using thiol poly (ethylene glycol) amine (SH-PEG-NH) as a spacer unit. Then, it was applied to selective capture of Aβ, and the resulting complex was directly analyzed by surface-assisted laser desorption ionization mass spectrometry (SALDI-MS). The results revealed that the Apt-GO could enhance the detection specificity and reduce non-specific adsorption. This method was validated to be sensitive in detecting Aβ at a low level in human serum (ca. 0.1 μM) within a linear range from 0.1 to 10 μM. The immobilizing amount of aptamer on the GO was calculated to be 36.1 nmol/mg (RSD = 11.5%). In conclusion, this Apt-GO-based SALDI-MS method was sensitive and efficient in selective extraction and detection of Aβ, which proved to be a good option for early AD diagnosis.