A simple schematic on parallel optical detection of two-fluorophore barcode for single-molecule nanopore sensing is presented. The chosen two fluorophores, ATTO-532 and DY-521-XL, emitting in well-separated spectrum range can be excited at the same wavelength. A beam splitter was employed to separate signals from the two fluorophores and guide them to the same CCD camera. Based on a conventional microscope, sources of background in the nanopore sensing system, including membranes, compounds in buffer solution, and a detection cell was characterized. By photoluminescence excitation measurements, it turned out that silicon membrane has a negligible photoluminescence under the examined excitation from 440 nm to 560 nm, in comparison with a silicon nitrite membrane. Further, background signals from the detection cell were suppressed. Brownian motion of 450 bps DNA labelled with single ATTO-532 or DY-521-XL was successfully recorded by our optical system.