The supramolecular oligomers play an important role because their preorganized precise structures lead to a well-defined structure–property relationship. Here, we employed an α-hemolysin nanopore as a single molecule tool to investigate the self-assembly supramolecular oligomers of bis(p-sulfonatocalix[4]arene) (bisSC4) and bis(methylviologen) (bisMV4+). The self-assembly of supramolecular oligomers could be dynamic monitored at single molecule level. The association constant between bisSC4 and bisMV4+ was calculated based on current blockage frequencies at the single molecule level. This work provides a complementary method to characterize the self-assembly oligomers with low molecular weight in solution.