In this paper, the ultrasensitive detection of an alpha-fetoprotein (AFP) is performed using a combination of two-dimensional (2D) surface-enhanced Raman scattering (SERS) and an AFP aptamer. We construct SERS "hot spots" through complementary base-pairing between substrates modified with AFP aptamer and silver nanoparticles modified with aptamer complementary sequence. The complementary adaptor sequence is also modified using Raman signal labeled molecule ROX, but the addition of the AFP damages the structure of nano-gap "hot spots", resulting in a decrease in intensity of the SERS signal. The ultrasensitive quantitative detection of AFP is realized using the changed working curve of the SERS signal of ROX. The detection limit of this method is 145 fg/mL, which is one order of magnitude higher than that of the traditional clinical detection method. The designed AFP SERS probe also exhibits good specificity and anti-interference ability. These results show that this new approach may provide a rapid and effective method for the accurate detection of AFP.