Modern molecular-biology applications raise renewed interest in sizing minute-amounts of DNA. In this work we utilize single-molecule imaging with in situ size calibration to accurately analyze the size and mass distribution of DNA samples. We exploit the correlation between DNA length and its fluorescence intensity after staining in order to assess the length of individual DNA fragments by fluorescence microscopy. Synthetic reference DNA standards are added to the investigated sample before staining and serve as internal size calibrators, supporting a robust assay for accurate DNA sizing. Our results demonstrate the ability to reconstruct the exact length distribution in a complex DNA sample by sizing a subset containing only femtogram amounts of DNA, thus, outperforming microfluidic gel electrophoresis which is the currently accepted gold standard. This assay may find useful applications for genetic analysis where the exact size distribution of DNA molecules is critical and the availability of genetic material is limited.