In this paper, impact of carbon on threshold voltage in MOSFET-based device is studied by 3D-atom probe tomography (APT). Carbon is one of most difficult contaminants incorporated from fab-environment to be detected by typical analytical techniques such as TEM-EDS or SIMS. Here, we successfully demonstrated the detection of carbon segregated at gate oxide/Si substrate interface using 3D-APT with single-atom sensitivity and sub-nanometer spatial resolution. It was found that the carbon contaminants have significant effect on the threshold voltage shift (ΔVth), in which ΔVth increases slightly with increasing carbon concentration. The deterioration of device performance is explained by means of which the positively ionized carbons at the interface acting as additional positive charges affecting the inversion to n-channel.