Optical design for multicolor fluorescence detection is the core part of multiplex quantitative polymerase chain reaction (PCR) system. According to the system requirements for excitation uniform, detection time and sensitivity, an optical system is presented, which is based on magneto-optical switch and photomultiplier tube (PMT) detector. Four LEDs excitation unit and PMT detection unit are adopted to improve system sensitivity. Electronic optical switch combined with the one-dimensional scanning mechanism and filter to implement four-color fluorescence scanning on a 96-well standard PCR plate. The design avoids the crosstalk among the four color optical paths. Whereas the crosstalk generated by the spectral characteristics of the fluorescent dye itself can be estimated by standard iterative four-dimensional clustering analysis algorithm. We finally evaluate the systematical fluorescent spectra crosstalk by the experimental study of four commonly used dyes.