Scanning force microscopy (SFM) reveals surface topography by scanning a sharp tip in close proximity to the sample. Due to tip–sample interaction, artificial broadening of the real surface structure with the tip geometry occurs. One approach for image reconstruction is the use of calibration standards, preferably in the size range of the samples. In the present study, an image reconstruction method based on colloidal gold as a geometric standard was used to reconstruct SFM images of biomolecules. Sample and calibration standard size were in the nanometer range, and the standards were coadsorbed with the specimen. Raw and reconstructed images of the biomolecules were compared, and the reconstruction was characterized by difference images as well as determination of the difference volume. The application of image reconstruction based on colloidal gold as a calibration standard for SFM of biomolecules is discussed.