We used high-resolution Kelvin probe force microscopy (KPFM) to investigate the immobilization of a prostate specific antigen (PSA) antibody by measuring the surface potential (SP) on a MoS2 surface over an extensive concentration range (1 pg/ml–100 μg/ml). After PSA antibody immobilization, we demonstrated that the SP on the MoS2 surface characterized by KPFM strongly correlated to the electrical signal of a MoS2 bioFET. This demonstration can not only be used to optimize the immobilization conditions for captured molecules, but can also be applied as a diagnostic tool to complement the electrical detection of a MoS2 FET biosensor.