Photon-counting analysis of nucleic acids plays a key role in many diagnostics applications for its accurate and non-invasive nature. However, conventional photon-counting instrumentations are bulky and expensive due to the use of conventional optics and a lack of optimization of electronics. In this paper, we present a portable, low-cost time-correlated single photon-counting (TCSPC) analysis system for DNA detection. Both optical and electronic subsystems are carefully designed to provide effective emission filtering and size reduction, delivering good DNA detection and fluorescence lifetime extraction performance. DNA detection has been verified by fluorescence lifetime measurements of a V-carbazole conjugated fluorophore lifetime bioassay. The time-to-digital module of the proposed TCSPC system achieves a full width at half maximum (FWHM) timing resolution from 121 to 145 ps and a differential non-linearity (DNL) between −8.5% and +9.7% of the least significant bit (LSB) within the 500 ns full-scale range (FSR). With a detection limit of 6.25 nM and a dynamic range of 6.8 ns, the proposed TCSPC system demonstrates the enabling technology for rapid, point-of-care DNA diagnostics.