The investigation of abnormal experimental phenomena observed in nanopore research improves our understanding of nanopores. In this article, we report and explore the unusual phenomenon that the amplitude of current blockage decreases beyond zero baseline (overflow amplitudes), which was observed in the translocation behavior of 100 bp double-stranded DNA molecules through SiNx nanopores. In our experiments, the overflow amplitude decreases with the increase of salt concentration and also decreases when the dwell time is shortened as the normalized amplitude of the overflow current showed a reduction with the increase of voltage. Upon analyzing the electric double layer meanwhile, the overflow amplitudes were shown to be positively correlated with the depth of the electric double layer and the duration of interaction between biological molecules. The formation of overflow amplitude can be attributed to the double electric layer ionic perturbation and reconfiguration, which are the results of the interaction between the biomolecule and the electric bilayer. The validation of the assumption using biomolecules containing different charges demonstrated that the overflow amplitude increased with the increase of the charge. It was concluded that proteins that pass through the nanopore with different orientation were differentiated based on their different overflow amplitude patterns. The investigation of overflow amplitude helps to enhance the understanding and the performance of nanopores.