Nanopore sensor conceptually represents an ideal single molecule counting device due to its unique partitioning-free, label-free electronic sensing. Existing theories and experiments have shown that sample concentration is proportional to the molecule translocation rate. However, a detailed nanopore geometry and size characterization or a calibration curve of concentration standards are often required for quantifying the unknown sample. In this work, we proposed and validated a calibration-free nanopore single molecule digital counting method for isolated molecule quantification. With the background ions as the in-situ references, the molecule translocation rates can be normalized to the ion translocation rates (baseline current). This in-situ reference alleviates the requirement for knowing the nanopore geometry and size or generating a calibration curve. In recognition of this effect, we developed a quantitative model for molecule quantification without the need for prior knowledge of experimental conditions such as nanopore geometry, size, and applied voltage. This model was experimentally validated for different nanopores and DNA molecules with different sizes. We anticipate this calibration-free digital counting approach would provide a new avenue for nanopore-based molecule sensing.