The invention discloses a multi-color super-resolution microscopy system and method based on a single channel. The system comprises a loss light source, a first collimating unit, a phase plate, a first excitation light source, a first dichroic mirror, a second collimating unit, a half wave plate, a polarizing beam splitter, a second excitation light source, a first detector, a second dichroic mirror, a lens-aperture-lens group, a third dichroic mirror, a scanning mirror, a second detector, a third collimating unit, a quarter wave plate, a microscopic objective and a sample stage. According to the system and method, two-color (or multi-color) super-resolution microscopy can be realized with one loss light source, and the system is simple in architecture and lower in cost. Stronger background noise caused by large energy consumption and crosstalk in a conventional single-channel multi-color super-resolution system can be overcome, the work precision is high, and the result is accurate. Therefore, compared with the prior art, the work efficiency and the experiment precision of the multi-color super-resolution microscopy system can be improved, and the cost is effectively reduced.