The authors describe a rapid and direct SERS-based immunoassay for the determination of AFP, an important marker for diagnosis of hepatocellular carcinoma. Silver nanoparticles (AgNPs; 36 nm i.d.) serve as a support to immobilize antibody and as a SERS intensifier, and AFP-modified gold nanoparticles are employed as capturing substrate. Direct and quantitative detection of AFP is accomplished with a limit detection as low as 5 ng·mL−1. Compared to assays based on the use of metal nanoparticles, the use of gold-silver nanoparticle heterodimers as an active SERS substrate can save costs because only a single antibody is required. Moreover, the high selectivity and good linear relationship of detecting AFP in fetal bovine serum indicates its potential applicability for the direct analysis of clinical samples.