To identify the extreme low concentration of alpha-fetoprotein (AFP) antigen in human serum for early detection of hepatocellular carcinoma, we aim to develop a high-sensitivity and low-cost AFP biosensor using quartz crystal microbalance (QCM) and cystamine self-assembly monolayer (SAM) technologies. In this study, the surface topographies of concentrations (0.1 and ) of AFP antibody with and without 1.25% glutaraldehyde cross-linking layer will be analyzed by an atomic force microscope system to investigate the effects of the glutaraldehyde layer on the sensing characteristics of the QCM-based AFP biosensor. According to our experimental results, the mass sensitivity was improved almost doubly (from ) as the glutaraldehyde layer was added between the cystamine SAM and the low-concentration AFP antibody. Either with or without the glutaraldehyde layer, higher mass sensitivity was obtained as the AFP antibody concentration increased to . However, a large AFP antibody requirement will increase fabrication cost and limit disposable application. We also demonstrated that high sensing linearities (95.67 to 99.6%) of the QCM-AFP biosensors can be achieved without being obviously affected by the glutaraldehyde layer and AFP antibody concentration.