Aiming at the crosstalk problem caused by small spectral intervals between fluorescent reagents in fourplex fluorescence quantitative polymerase chain reaction (PCR) detection analysis system, we calculate and analyse the effect of cut-off steepness, central wavelength positioning, and bandwidth of filters on crosstalk. Design and prepare four sets of fluorescence excitation and emission filters with proper shape cut-off steepness (optical density (OD) from OD 0.3 to OD 6 is less than 8 nm) and bandwidth (9–11 nm). In fourplex PCR instrument, crosstalk coefficient in all four channels are less than 0.3%.