With the continuous demand for technological advances in nucleic acid detection for use in medical testing, point-of-care testing (POCT) has emerged as a research focus, presenting various challenges to overcome. To circumvent the disadvantages of POCT nucleic acid detection instruments that are currently in use, a straight-axis multi-channel optical detection system is proposed and designed, which facilitates both real-time, multi-channel as well as instant detection of nucleic acids . First, an optical path is built based on an orthogonal optical path design. Second, a new straight-axis multi-channel integration and switching device is designed to enable real-time switching between multiple fluorescence detection channels. Finally, the effectiveness of the optical path design is verified by simulation; this system is integrated into a self-designed polymerase chain reaction thermocycler, and a real-time fluorescence detection experiment is conducted using a new coronavirus standard. This system reaches a minimum coefficient of variation value of 0.02%. The minimum channel resolution is 0.49 mg/mL, and no channel crosstalk is observed. The system demonstrates good on-site detection capabilities, and functions cooperatively with the instrument to complete on-site detection from “sample in” to “result out”, offering a new perspective for POCT using multi-channel real-time fluorescence detection of nucleic acids.