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We report, for the first time, the use of single poly (methyl-methacrylate) (PMMA) polymer-based nanopores to not only detect the translocating biomolecules of dsDNA and BSA in individual populations, but also distinguish between them in a mixed population, based on the obtained depths (iBlock) and durations (tD) of the ionic current blockade events. Quantitative analyzes of current blockade events induced by separate populations of dsDNA and BSA molecules translocating through single PMMA-based nanopores revealed a mean iBlock value of 22.4 ± 0.69 pA with a tD of 0.202 ± 0.009 ms, and a mean iBlock value of 60.14 ± 0.19 pA with a tD of 0.59 ± 0.08 ms for dsDNA and BSA molecules, respectively. Evaluation of translocation events by dsDNA and BSA molecules in a mixed population through single nanopores indicated two distinct groups of current blockade events, extrapolation of which could perfectly correlate the group demonstrating iBlock values of 13-38 pA to the dsDNA molecules, whereas the group demonstrating iBlock values of 52-73 pA to the BSA protein molecules transporting through the nanopores. Analysis of the data suggests that the majority of dsDNA or BSA protein molecules translocating through the nanopores are in their partially folded conformation. This paper is an important milestone in the development of polymer-based solid-state nanopore devices for fast and accurate biomolecule detection, differentiation/discrimination, and future structural characterization.