The most commonly applied method of fluorescence detection for on-chip gel electrophoresis is finish line detection in epifluorescence configuration using highly sensitive photodiode or a cooled scientific camera as a photodetector. In this paper we aim to confirm that a simple optical configuration based on a limited number of components and a low-cost, non-cooled CCD matrix detector combined with software image processing makes possible very sensitive fluorescence detection for high resolution for on-chip gel electrophoresis. The position and dimensions of software defined detection areas (so called regions of interest) have been identified as the main factors influencing detection properties. Several parameters which typify electropherograms (including signal-to-noise ratio and full width at half maximum of the peaks) have been studied. Finally, the developed detection unit was used in the separation of a DNA ladder with a theoretical number of over 600,000 of separation plates. Thus, the applicability of the detection unit has been confirmed. (C) 2016 Elsevier B.V. All rights reserved.